Study On Vector-based Double-stranded Oligonucleiotide In Transcriptional Factor Decoy Strategy

Transcriptional factor decoy strategy (TFD strategy), a strategy in which the oligodeoxynucleotide (ODN) plays a key role by specific sequences they contain, has been widely applied in investigation of bioinformation about transcriptional factors and other molecules. It is the competition between exogenous and endogenous cis-elements for combination with transcriptional factors that assigns ODNs the ability to inhibit the activities of transcriptional factors because of the exogenous cis-elements they contain. This novel strategy has been applied in studies of many transcriptional factors, such as NFκB, Sp1, NFAT, etc. As a novel pharmacotherapy treatment with great potential, TFD strategy has been used in vivo and initial clinical experiments, encouraging results obtaining.Although there are so many advantages when studying by TFD strategy, some defects cannot be disregarded, in which the pool transferring efficiency into cell nuclears of ODNs is one of the most one. When transferred into cells, almost 90% of ODNs (without any modification) will be uptaked and degenerated by cytolysosome, meaning only 10% of ODNs can go into cell nuclears and fulfil their duty, which shortcoming restricts function of TFD strategy severely. Up to now several settlements have been used to overcome this difficuty, including crosslinking of nuclear localization signal peptide (NLS) to the end of ODN, modification of ODN for increasing resistance of ODN to cytolysosome and the application of novel transfecting agents for better transfecting efficiency. However, the high cost or the complicated technology prevents these methods from the application on improvement of TFD strategy. Thus new, convenient and economic methods should be found.In our previous comparison between TFD strategy and RNAi, we found that there will be a step producting local double-stranded RNA in the process of expressing of siRNA by plasmid vectors. What we want to know is whether this kind of dsRNA, containing sequences as cis-elements except a replacement of T by U, could block transcriptional factors as ODNs with the same cis-elements. If that is true, the transferring efficiency could be conspicuously increased when plasmid expressing this kind of dsRNA packed with virus, or virus vectors used directly for expressing dsRNAs. This is what we hope to demonstrate in this report.【Objectives】(1) To determine whether vectors expressing dsRNAs containing sequences similar to cis-element of some transcriptional factor (except a replacement of T by U) could inhibit the transcriptional activity of this transcriptional factor; (2) To investigate the mechanism of the inhibiting effect.【Methods】(1) NFκB, a transcriptional factor expressing in HEK293 cells, was chosen and related vectors had been constructed, including vector-based ON (V-B ON, vector expressing dsRNA containing sequence similar to a cis-element of NFκB) and its scrambled control vector; shRNA of NFκB (positive controlⅠ). oligo-ODN without any modification (containing the same cis-element) and its scrambled control had been synthesized also, the former naming positive controlⅡ. (2) These vectors or fragments were transfected into HEK293 cells and MTT assays were carried out to detect the growing change of treated cells; flow cytometry was carried out to detect apoptosis of transfected cells after treated with DNA damaging agent; quantitative real-time PCR and luciferase reporter gene analysis were used to determine the changes of transcriptional activities of IL-8 and MCP-1, two downsteam genes of NFκB; (3) FOS, AP-2αand RXRA, three transcriptional factors expressing in HEK293 cells, were chosen randomly to determine whether this kind of inhibiting effect was pervasive by detecting transcriptional activities of the downstream genes of the four transcriptional factors (NFκB, FOS, AP-2αand RXRA) by quantitative real-time PCR; (4) Transcriptional activities of this four transcriptional factors were detected by quantitative real-time PCR to determine whether this kind of inhibiting effect was due to RNAi effect of vectors.【Results】1.V-B ON could inhibited the growth and promoted apoptosis of treated HEK293 cellsVarious vectors were constructed successfully and transfected into HEK293 cells, oligo-ODN and its scrambled control as well. Time-course experiments by MTT assay were carried out at 24H, 48H and 72H post-transfection. The results showed that there was a tendency in all of these three time courses that the relative inhibiting ratios to cell growth of V-B ON and shRNA were similar (V-B ON:42.3±0.152%, 57.2±0.612%, 29.1±0.441%; shRNA: 54.2±0.231%, 64.3±0.246%, 31.6±0.225%), except that of oligo-ODN ,which were relatively lower (ODN: 18.2±0.294%, 21.1±0.546%, 11.6±0.331%).According to the results of MTT assays, 48H was chosen to be the time for subsequent experiments. 24 hours after transfection, cells were treated with Etoposide (50uM) for 24H. Then cells were collected for flow cytometry. The results showed that V-B ON and shRNA performed similar effect on promoting cell apoptosis (V-B ON: 42.2±0.437% vs shRNA: 50.2±0.512%), which was more powerful than that performed by oligo-ODN (22.3±0.601%). The relative inhibiting ratios mentioned above were ratios of vectors / fragments compared with each negative control (V-B ON vs scrambled V-B ON; shRNA vs pTER+; ODN vs scrambled ODN).2. V-B ON could affect the transcription of downstream genes of transcriptional factors in transfected cells.Luciferase reporter gene vectors of IL-8 and MCP-1,two downstream genes of NFκB, were constructed and applied to detect the transcriptional activities of IL-8 and MCP-1 in transfected cells. The reporter gene analysis showed that the transcriptional activities of these two genes were both blocked in cells transfected with V-B ON, just like the effect of shRNA and oligo-ODN.FOS, AP-2αand RXRA, three transcriptional factors expressing in HEK293 cells, were chosen randomly and related vectors were constructed. Vectors and fragments were transfected into cells respectively, followed with RNA extraction, reverse transcription and quantitative real-time PCR. The results showed that there was a down-regulation of transcriptional activities of downstream genes of these four transcriptional factors (up-regulation if the expression inhibited by its upstream factor). V-B ON exhibited similar inhibiting effect as shRNA, which was more powerful than that of oligo-ODN. 3. V-B ON could not affect the transcriptional activities of corresponding transcriptional factor in transfected cells, which demonstrated that at least V-B ON would not show its power through RNA interference against corresponding factor.RNA was extracted from transfected cells and quantitative real-time PCR was performed to detect mRNA expression level of these four transcriptional factors themselves. The results showed that V-B ON did not showed capability to inhibit transcriptional activities of corresponding factors, compared with the noticeable decrease of activities caused by shRNA.【Conclusions】In this report we for the first time raise a hypothesis that vector expressing double-stranded RNA containing sequence similar to cis-element of some transcriptional factor could show similar inhibiting effect against the factor as ODN in Transcriptional Factor Decoy strategy. Evidences about inhibiting capability of this kind of vector were provided according to our experiments and mechanisms were investigated partially. This kind of vector might perform a ODN-like capability through double-stranded RNA containing sequence similar to cis-element of transcriptional factor (T→U). These findings may be helpful for the deeper cognition to RNAi and TFD strategy, and provide a useful tool for molecular biological investigation.