Investigate The Biogenesis Of CML With Small RNA And RNAi

CML is a disease that results from a chromosomal translocation in hematopoietic stem cells (HSC), Bcr-Abl and p210 Bcr-Abl have been proven to cause CML and used as a target molecule for selective antileukemia therapy. But, the biogenesis of disease is a process involved in many gene, and Bcr-Abl how causes the clinical CML syndrome is still unclear. To identify more potential targets, we used RNA interference (RNAi) as a tool for the biogenesis of CML and establish a relationship of CML and miRNAHere, Bcr-Abl,Abl,Erk1,Gab2 and Lyn were choosed to study. After expression of these genes were investigated by RT-PCR, The small interference RNA ( siRNA) was synthesized in vitro. Then K562 cells stably expressing Bcr-Abl gene were transfected with the siRNA by lipofectamine in different parameters, inhibitory effect of siRNA was demonstrated by semi-quantitative RT-PCR. Results demonstrate: siRNA could inhibit Bcr-Abl and Abl to 100% at 24 h and 48 h after transfection, the mRNA level Erk1 and Gab2 were suppressed to 100% and 42% at 24 h respectively, however Lyn has no change. When increase cell number, the tendency of inhibition keep in line and show a relationship with siRNA absolute dose. All suggest these genes have dependability on transcription or post-transcription. Design and effective condensation of siRNA are two important parameter during RNAi.Combined bioinformatics and the new theory on miRNA, the cause of foregoing phenomenon was analyzed. When promoter sequences of these genes were aligned by Clustw program, homological sequence is about 250 bp between Bcr-Abl,Gab2 and Erk1. then the structure of 269 bp region in Gab2 was predicted by mfold, apparent character with pre-miRNA was displayed between -80 to -150 region, 30 nt loop,21 and 17 nt arm. To accord with mature miRNA, 21 nt arm sequence was used for following study.After synthesized oligonucleotide probe and enriched small RNA with miRNA extraction kit, northern blot was performed. By optimized probe labeling, hybridization,washing conditions and image formation, positive band of pre-miRNA was observed. To acquire mature miRNA and guarantee stability of result, manipulation is need to optimized.To investigate the potential function of this miRNA, After being synthesized and annealed,the shRNA according to miRNA was inserted into a expression vector (RNAi-Ready pSIREN-RetroQ-ZsGreen) driven by U6 promotor. The positive plasmids were sequenced and extracted by Endo-free Plasmid Maxi Kit. Because the efficency of transient transfection can not satisfied for functional assay, a positive cell clone should be established for long-term research.In this paper,depending on RNAi technique,the relationship of homologous gene on transcription or post-transcription was determined. Then, combined the thinking of miRNA regulation, the phenomenon was analyzed and get preliminary result. This research provided new exploration for investigating the biogenesis of CML and develope gene therapy.