Abstract
Purpose: Human telomerase reverse transcriptase (hTERT) plays a crucial role in tumorigenesis and progression of cancers. Gene silencing of hTERT by short interfering RNA (siRNA) is considered as a promising strategy for cancer gene therapy. Various algorithms have been devised for designing a high efficient siRNA which is a significant issue in the clinical usage. Thereby, in the present study, the relation of siRNA designing criteria and the gene silencing efficiency was evaluated.Methods: The siRNA sequences were designed and characterized by using on line soft wares. Cationic co-polymer (polyethylene glycol-g-polyethylene imine (PEG-g-PEI)) was used for the construction of polyelectrolyte complexes (PECs) containing siRNAs. The cellular uptake of the PECs was evaluated. The gene silencing efficiency of different siRNA sequences was investigated and the effect of observing the rational designing on the functionality of siRNAs was assessed.Results: The size of PEG-g-PEI siRNA with N/P (Nitrogen/Phosphate) ratio of 2.5 was 114 ± 0.645 nm. The transfection efficiency of PECs was desirable (95.5% ± 2.4%.). The results of Real-Time PCR showed that main sequence (MS) reduced the hTERT expression up to 90% and control positive sequence (CPS) up to 63%. These findings demonstrated that the accessibility to the target site has priority than the other criteria such as sequence preferences and thermodynamic features.Conclusion: siRNA opens a hopeful window in cancer therapy which provides a convenient and tolerable therapeutic approach. Thereby, using the set of criteria and rational algorithms in the designing of siRNA remarkably affect the gene silencing efficiency.
Highlights
The Human telomerase reverse transcriptase (hTERT) gene is a key component of the telomerase catalytic activity, which encodes the reverse transcriptase part of the telomerase complex.[1]
Targeting 3′untranslated region (UTR) of mRNA by complementary short interfering RNA (siRNA) leads to translational repression which mediated by Ago[1], Ago[3] and Ago4.7,8 The efficiency of siRNA is dependent on various factors including sequence space, target availability, the position of nucleotides, secondary structures of mRNA and intrinsic characteristics of siRNA and target mRNA
Conclusion siRNA-based gene therapies are emerging as a promising novel therapeutic approach for the treatment of various diseases such as cancer
Summary
The hTERT (human telomerase reverse transcriptase) gene is a key component of the telomerase catalytic activity, which encodes the reverse transcriptase part of the telomerase complex.[1]. RNA interference (RNAi) is a sequence-specific gene silencing system which knocks down the gene expression by using double-stranded RNA (dsRNA) homologous to the target gene.[3] The capability of siRNA to induce gene silencing introduces this mechanism as a powerful functional genomic tool.[4] The gene silencing effects of RNAi can be exerted by two different mechanisms. The RISC activation induces different mechanisms varying from the repression of translation to the degradation of mRNA which is dependent on the target site of mRNA. Targeting 3′untranslated region (UTR) of mRNA by complementary siRNA leads to translational repression which mediated by Ago[1], Ago[3] and Ago4.7,8 The efficiency of siRNA is dependent on various factors including sequence space, target availability, the position of nucleotides, secondary structures of mRNA and intrinsic characteristics of siRNA and target mRNA. Regions of the gene sequence represents less than 78% query coverage with other genes may be a desirable sequence.[17]
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