Designing and development of a silencer vector based on second generation of RNA interference [RNAi] technique to study downregulation of steroid receptor RNA activator [SRA]

ABSTRACT

RNA interference [RNAi] is the most potent technique for gene silencing in eukaryotic cellular system at transcriptomic level. Genetic disorders and cancers are important targets for therapeutic development of this technique. In order to bypass the temporary dpwnregulation by siRNA, a new generation of shRNA named shRNAmir has developed. Silencing construct with structure similar to microRNA [shRNAmir], mimics a natural microRNA pathway inside the cell. Steroid receptor RNA activator [SRA] is one of the regulators of steroid receptor like ER. Prostate, uterus and breast tissue express a low level of SRA, there is an increase of expression during their tumorgenesis. So SRA may participate in tumorgenesis or proliferation of tumors. We used RNAi technique to silence expression of SRA. The SRA silencer was designed and constructed by Soe-PCR, then cloned into an expression vector pEGFPCl. Human breast cancer [MCF7] cells were transfected with silencer plasmid then the changes in the SRA expression estimated by Real-Time PCR at 24, 72 hours and after l0days. The results showed about 60% decrease in relative expression of SRA gene, after 72 hours and 10 days, which shows that shRNAmir-SRA could successfully knockdown the expression of target gene. It seems that the designed shRNAmir may be a suitable tool for a variety of applications because it could stably knockdown the expression of target gene