A Study On The Mechanism Of High-dose EsiRNA Induced Adar-1 Gene Transcriptional Upregulation

RNA interference is a post transcriptional gene silencing mechanism conserved in evolotion from warm to human which has been used as a powerful tool for gene fucntional research.With its great development,many research groups have applied RNAi into gene function investigation, signal transduction researh and exploration of the initial mechanisms of diseases.Although a few proteins involved in RNAi pathway has been identified,the detail of regulation mechanism of RNAi is still in the dark.Data from my colleagues has proved that,high-dose esiRNAs,when enter mammalian cells,will lead to increase in adar-1 mRNA,which further suppresses the efficacy of esiRNA.Meanwhile,when esiRNAs of adar-1 and eri-1 were introduced into cells with target gene esiRNA,we observed enforced gene silencing efficiency.This observation causes our concern on the promoter region of adar-1.In this dissertation,we report our priliminary investigation of adar-1 promoter responsing to high-dose non-specific esiRNA.In this part of work,we replaced CMV promoter of plasmid pEGFP-N1 and pSEAP2-control with truncated adar-1 promoter region of different lengths, and co-transfected them into CHO cells with high-dose non-specific esiRNA or control dsDNA.By observing the intensity of green fluorescence and absorbance of SEAP at 405 wavelength,we identified the shortest section of adar-1 promoter which can response to high-dose esiRNA induction.The result shows that the shortest section of adar-1 promoter which could response to high-dose esiRNA is 200bp upstream of ATG initiation codon. To further determine the response elment,we search this 200bp sequence in online database and identified PU.1 binding site as a candidate element. Then,we adopted deletion mutation to study the role of PU.1 binding site in the esiRNA induced adar-1 transcription up-regulation.The data demonstrate that the deletion of PU.1 binding site abolished adar-1 promoter responsing to high-dose esiRNA induction.The intensity of green florescence and secreted alkaline phosphatase showed no difference with control groups.When this element was added to the 180bp region upstream ATG codon,the response to high-dose esiRNA was restored.This implied that PU.1 binding site plays a key role in the high-dose esiRNA induced adar-1 transcription up-regulation.To determine the function of transcription factor PU.1 in this high-dose esiRNA induction,we transfected specific PU.1 esiRNA into CHO cells to silence endogenous PU.1 gene,and then observed the increase in reporter gene transcription induced by high-dose esiRNA.We discovered that,after endogenous PU.1 was knock down,high-dose esiRNA could no longer induce increase of adar-1 transcription,the experimental goup shows no remarkble fluorescent enhance comparing to the contrl group.Also,when overexpressing PU.1 in CHO cell,which was to mimic the condition of high-dose esiRNA induction,intensity of secreted alkaline phosphatase was comparable to high-dose esiRNA transfected group.These data further conformed that PU.1 is involved in the high-dose esiRNA induced adar-1 transcription up-regulation,thus affects the efficiency of RNA interference.