Abstract
RNA interference (RNAi)/miRNA has been recognized as one of the most important regulation mechanisms of gene expression at posttranscriptional levels in eukaryotic cells. Although the main components within the RNAi/ miRNA pathway have been identified and characterized, studies on the molecular mechanisms regulating the activity of the RNAi/miRNA pathway have begun to emerge in recent years. High-throughput reporter assays have been developed to monitor the activity of the RNAi/miRNA pathway and applied for proof-of-concept pilot screening, leading to identification of some inhibitors and activators that are generally or specifically regulate activity of the RNAi/ miRNA pathway. Additionally, in combination with multidisciplinary approaches such as proteomics, biochemistry and genetics, to identify the targeting components, some protein co-factors that to play significant roles in the regulation of the activity of the RNAi/miRNA pathway have been identified. Herein we highlight the high throughput reporter assays developed in recent years and the resulting discovery of the RNAi/miRNA enhancers and inhibitors, with attention on developing novel RNAi- or miRNA-based therapeutic interventions.
Highlights
RNA interference (RNAi) has been recognized as one of the most important mechanisms at post transcriptional level in eukaryotic cells, mediated by small non-coding RNA, including in particular, interference RNA, microRNAs and piwi-interacting RNA with length of 18-30 nt, to regulate diverse metabolic pathways involved in chromosome architecture and segregation behavior, transcription, and RNA processing and stability, and thereby to phenoltypically coordinate development, growth control, apoptosis, self defense, and stem cell [1,2,3,4,5,6]
Main components of the RNAi/miRNA pathway have been identified and characterized [2], studies on the regulation mechanism of the pathway itself have received high attention in recent years by using the chemical biology approach, which offers a more dynamic way to monitor the activity of specific pathways compared to traditional forward or reverse genetic approach
The emergence of the chemical biology approach to the RNAi/miRNA pathway is still in its infancy, the approaches led to further understanding on the regulation mechanisms by identification of a number of small molecule modulators targeting to the specific steps in the RNAi/miRNA pathway
Summary
RNA interference (RNAi) has been recognized as one of the most important mechanisms at post transcriptional level in eukaryotic cells, mediated by small non-coding RNA, including in particular, interference RNA (siRNAs), microRNAs (miRNAs) and piwi-interacting RNA (piRNAs) with length of 18-30 nt, to regulate diverse metabolic pathways involved in chromosome architecture and segregation behavior, transcription, and RNA processing and stability, and thereby to phenoltypically coordinate development, growth control, apoptosis, self defense, and stem cell [1,2,3,4,5,6]. Given that it takes too long and costs too much to bring new drugs to market and that different targets of the compounds may bear unknown therapeutic functions, it is plausible to speculate screening existing drugs for new uses [29] as an example detailed below that a general antibiotic enoxacin has been proven to function as cancer cell growth inhibition For screening of such a large number of clinical drugs to dissect the RNAi/miRNA pathway, the reliable and robust assays fluorescence-based reporter system is indispensable. Clinically used for anti-bacterial therapy, has turned out to have a novel function as a anti-cancer agent, serving as example that old drugs might have new uses This assay is not biased towards or against any specific components of miRNA pathway and could confer high efficiency for high throughput screening of the small molecule library. It is still not a perfect report assay system in a way that it is unable to distinguish whether the decrease of EGFP levels is due to the drug toxicity-caused
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