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摘要
The Argonaute proteins play essential roles in development and cellular metabolism in many organisms, including plants, flies, worms, and mammals. Whereas in organisms such as Caenorhabditis elegans and Arabidopsis thaliana, creation of Argonaute mutant strains allowed the study of their biological functions, in mammals the application of this approach is limited by its difficulty and in the specific case of Ago2 gene, by the lethality of such mutation. Hence, in human cells, functional studies of Ago proteins relied on phenotypic suppression using small interfering RNA (siRNA) which involves Ago proteins and the RNA interference mechanism. This bears the danger of undesired or unknown interference effects which may lead to misleading results. Thus, alternative methods acting by different regulatory mechanisms would be advantageous in order to exclude unspecific effects. The knockdown may be achieved by using specific antisense oligonucleotides (asONs) which act via an RNase H-dependent mechanism, not thought to interfere with processes in which Agos are involved. Different functional observations in the use of siRNA versus asONs indicate the relevance of this assumption. We developed asONs specific for the four human Agos (hAgos) and compared their activities with those obtained by siRNA. We confirm that hAgo2 is involved in microRNA (miRNA)- and in siRNA-mediated silencing pathways, while the other hAgos play a role only in miRNA-based gene regulation. Using combinations of asONs we found that the simultaneous down-regulation of hAgo1, hAgo2, and hAgo4 led to the strongest decrease in miRNA activity, indicating a main role of these proteins.
- 出版日期2010-12
