Conditional Gene Silencing Using A Tumor-specific And Heat-inducible SiRNA System

RNAi is a highly conserved regulatory process that exerts the specific post-transcriptional gene silencing observed in a variety of species, and it has been already exploited as a powerful tool for genetic manipulation. It allows researchers to silence virtually any gene to elucidate molecular pathways and phenotype/genotype relationships. But there are many obstacles and concerns that prevent RNAi from being widely used in gene therapeutic field. Among them, the non-spatial and non-temporal control is the primary limitation, as well as off-target effects. Inducible RNAi technology can effectively regulate target gene by inducer mediated sh RNA expression. Combination with inducible regulation system makes RNAi technology more sophisticated and may provide a wide application field. In this study, we inserted appropriate heat shock elements into tumor specific promoter sequences, which aimed to construst a temperature-inducible RNAi system. The main study and results are summarized as follow:① h TERT is highly expressed in tumor cells, but not in normal cells. The heat-shock response depends on the effective combination of HSE and HSF. Here we select three kinds of HSE: the first kind of HSE is derived from Hsp27 that belongs to small HSP. The other kind is made of repeat arrangement of the three HSE basic units “NGAAN-NTTC”. The last one is composed of the “AGAAC”and “GTTCT” motifs, but with reversed orientation of three of the motifs. HSE is inserted into the-65 and-85 site of h TERT promoter, as well as the-222 site.② Construct the ph TERTp-EGFP、ph TERT/HSE(AB)p-EGFP、ph TERT/HSE(MN)p-EGFP and ph TERT/HSE(XY)p-EGFP plasmids. We determined the driving ability of different promoters by detecting the protein expression level of EGFP in breast cancer cells MCF-7. Results show that, compared with h TERT, h TERT/HSE(MN) and h TERT/HSE(XY) promoters not only can reduce the local expression level but also response to hyperthermia effectively. In addition, these promoters presented a lower EGFP expression level than Hsp70 in Ha Ca T and HL7702 human normal cells, which suggests the ph TERTp-EGFP and ph TERT/HSEp-EGFP plasmids have good tumor specificity.③ Construct the p Mh TERTpsi SNCG and p Mh TERT/HSEpsi SNCG plasmids. From the expression level of RNA and protein, we determined the efficiency of the inducible si RNA system by targeting SNCG gene in MCF-7 and Hep G2 cells. Results show that the controllable si RNA system can be induced to initiate si RNA expression by heat-induce. The silencing effect of SNCG is on a relative low level(10%) at 37℃ while it is significantly increased to 50% or 60% after heat inducing at 43℃.