Effect Of ShRNA Expression On Intracellular MiRNA And Growth Factor

RNA interference (RNAi) is a gene silencing phenomenon which was first discovered in the C.elegans triggered by double-stranded RNA. Then subsequent studies confirmed that longdouble-stranded RNA (dsRNA) and small hairpin RNA (shRNA) would be cut into21ntfunctional double-stranded RNA--shRNA by Dice. Mature miRNA bound to proteinArgonaute(Ago) which caused target mRNA degradation. The shRNA and miRNA share thesimilar production pathways and have similar functions, the main difference lies in their differentsources: usually shRNA introduced in in vitro, while the miRNA encoded by the genome andtranscribed.RNAi technology has been widely used in the study of antiviral research with some significantprogress. However, the application of RNAi also encountered some challenges, such as off-targeteffects, non-specific immune response reaction, cellular pathway disorders and fatal toxic sideeffects. Off-target effects of RNAi existed widely in model organisms. The off target effects maylead the body some toxic phenotypes. Nevertheless, only limited sequence complementary pairing,shRNA can still silencing their cognate target gene or the same sequence gene. Another challengewill cause the interferon effect promoted by non-specific immune response due to the similarstructure of virus. Overexpressions of shRNA not only triggered saturation exogenous constructsand cause dysfunction of miRNA pathway but also cause the non-specific interferon response andeven cause the transgenic animals lethal.In order to explore the changes in expression of endogenous factors and early embryonicdevelopment duo to expression of siRNA in cells, we first use liposomal transfection and then use450μg/mL G418drug resistance screening and indirect fluorescence immunoassay (IFA) assay toobtain4species clone cells of different types that overexpression different types of shRNA and ablank plasmid control cells that transfect with scramble expression plasmid (shRNA-scramble).Extracts total RNA from the porcine fetal fibroblasts, which express different types of shRNA,using Realtime PCR method to detect cell response effect of endogenous gene IFN-β and OAS1,and the results show that IFN-β and OAS1mRNA expression were significantly increased (P<0.5), that high expression of shRNA induce cells to produce interferon response. While usingRealtime PCR to detect cutting tools of small RNA enzyme Dicer and Drosha mRNA, results show that the two tools enzyme mRNA expression levels was significantly increased (P <0.5).Extract small RNA from different types of porcine fetal fibroblasts, using Realtime PCR methodto detect the intracellular part of the miRNA and found that mir-21and mir-196expression wassignificantly increased (P <0.5), based on the high expression of miRNA and small RNA enzymeare elevated to the fact that overexpressiong of shRAN disrupted endogenous miRNApathway,whilenormal cell growth may be adversely affected.Then selected stably expressing thedifferent types of shRNA porcine fetal fibroblasts for somatic cell nuclear transfer, after72hourstransplantation statistical blastocyst formation rate was observed, the comparison found that therewas no significant difference among thedifferent shRNAcells. Indicating that early embryonicdevelopment is no obvious effect by shRNA phenotype.