The Effects Of Proliferation And Apoptosis In Acute Promyelocytic Leukemia Cell By Small Interfering RNAs

The aim of this study is to investigate the effects of small interference RNA(siRNA)targeting PML-RARa mRNA on proliferation inhibition and induction apoptosis in human Acute Promyelocytic Leukemia cell line NB4, and to provide the theoretical rationale and experimental evidence for its potential clinical application for anti-APL.The gene sequence of PML-RARa mRNA was found from the GeneBank. The target gene site on the PML-RARa mRNA were selected according to Max-Planck-Institute (MPI) and the rational siRNA design rules,the secondary structure of candidate targeted mRNA was predicted,the thermodynamic parameters of them were analyzed,the targeted gene sequences were compared with BLAST to emilinate any sequences with significant homology.The proliferation inhibition was evaluated by MTT assay and colony-formation inhibiting test.Apoptosis was determined by flow cytometry (FCM) and the morphology apoptotic cells was identified by Giemsa-Wright staining.Western blot was used to analyze the expressions of PML-RARa fusion protein of NB4cells after siRNA treatment.The mRNA local secondary structure caleulated by RNA structure software, and the optima design of specific siRNA contributed by bioinformatics rules.And then five sequences of PML-RARa siRNA have been designed and synthesized in vitro.The three sequences, siRNA1244,siRNA1246and siRNA1690which showed the most effective inhibition of NB4cell growth,were identified among the five candidate siRNAs,with cell proliferative inhibitory rate nearly50%after NB4cell exposed to12.5nmol/L-50nmol/L siRNA1244for24,48and72hours.The50%inhibitory concentrations(IC50)of siRNA1244,siRNA1246and siRNA1690for24h were46.578nmol/L,59.324nmol/L and62.620nmol/L, respectively;and65.668nmol/L,76.549nmol/L,74.430nmol/L for72h respectively.The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibitied.FCM results showed that the rate of cell apoptosis increased after transfecting siRNA24hours.The results of annexinV/PI staining detected cells apoptosis indicated that the rate of apoptosis improved(1.53%,15.26%,64.50%,57.53%and21.46%)following the augmentation of the different siRNA (siRNA30,siRNA382,siRNA1244,siRNA1246and siRNA1690for24h). Morpholog- ical analysis through microscopy showed the shrinking of cell volume and typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic body"after NB4cells exposure to siRNA. Western blot analysis showed that PML-RARa proteins reduced sharply after a single dose of50nmol/L siRNA transfeetion.The proliferation of NB4cell was remarkbly inhibited by siRNAs (siRNA1244, siRNA1246and siRNA1690) in a concentration-dependent manner in vitro.The siRNAs can effectively induces apoptosis at a concentration of50nmol/L. It is likely one of anti-leukemia mechanism in NB4cell treated with siRNA that PML-RARa targeted protein was highly down-regulated. The siRNAs (siRNA1244, siRNA1246and siRNA1690) was proved valuable in the treatment of APL.