RNAi Applications: Development of Approaches for Selection of siRNAs and Characterization of miRNA Function in Cancer.

摘要

RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression mainly through small interfering RNAs (siRNAs) and microRNAs (miRNAs). SiRNAs have revolutionized experimental biology and gained recognition as a powerful tool for functional genomics studies. The sequence-specific selectivity and robust capacity of siRNA to inactivate genes in vivo has also led to the development of novel RNAi-based therapeutics. Recently, miRNA has attracted considerable attention due to its important roles in the regulation of various cellular processes, especially its link with cancer. Understanding of RNAi has advanced remarkably worldwide. In light of such progress, this project aimed to develop a high throughput approach for forward screening of the most effective siRNAs in mammalian cells, and to comprehensively analyze sequence variations in both miRNA targets and miRNA genes in human tumor tissues and characterize their roles in tumorigenesis.;To investigate whether single-nucleotide polymorphisms (SNPs) located at miRNA-binding sites affect the expression of miRNA targets and thus contribute to oncogenesis as the second objective, we firstly performed a genome-wide analysis of the miRNA-binding SNPs at the 3' UTR of various human genes. We found that miRNA-binding SNPs are negatively selected with respect to SNP distribution between the miRNA-binding seed region and entire 3'-UTR sequences. We then compared the expression of each miRNA binding SNP in cancerous and normal tissues and found that the allele frequencies of some miRNA target SNPs significantly differ between human cancer EST libraries and the dbSNP database. Furthermore, we experimentally validated that the allele frequency of 12 miRNA-binding SNPs in human cancers is indeed aberrant. Hence, miRNA-binding SNPs affect miRNA target expression and function, and therefore are potentially associated with cancers.;To determine whether genetic alterations in miRNA genes are associated with cancers, we systematically screened sequence variations in several hundred human miRNAs isolated from about 150 tumor tissues and 20 cancer cell lines. We identified eight new SNPs and 14 novel mutations (or very rare SNPs) that specifically present in human cancers. While most of the mutations did not exert detectable effects on miRNA function, a G-to-A mutation in pre- let-7e significantly reduced its expression in vivo, indicating that a mutation of miRNA could contribute to tumorigenesis. These results suggested that further screening for genetic variations in miRNA genes from human cancers should increase the discovery and identification of molecular diagnostic and therapeutic targets and thus complement mutational analysis of consensus coding sequences in human cancers.;To develop a genetic forward approach for screening of siRNAs as the first objective, siRNA expression libraries were directly delivered into mammalian cells via bacterial invasion. The target genes were fused with the suicide gene, cytosine deaminase, and stably expressed in mammalian cells. The most effective siRNAs were conveniently and robustly identified in surviving cells under drug selection without the prior assay of the RNAi potency of individual siRNAs in all wells that is required by other siRNA screening methods. Significantly, the potent siRNAs isolated from the surviving cells proved to have noncellular toxicity. Hence, this approach can robustly enrich the most potent and nontoxic siRNAs in surviving cells, and thus has considerable potential to facilitate RNAi applications by minimizing the dose-dependent and sequence-nonspecific side effects of siRNAs.