論文の公開元へA Study of The Effects of Small Molecules on Dicer-mediated Cleavage of Precursor miRNAs (pre-miRNAs)
概要
MicroRNA (miRNA) is a small (18-22 nucleotides) and evolutionary conserved non- coding RNA that is expressed as a post-transcriptional regulator of living cells and is known to be implicated in various cellular processes and diseases. On the normal condition, miRNA was tightly regulated in the cells, however, the aberrant expression of this type of non-coding RNA would result in cancer, and this type of miRNA is commonly referred to as oncomirs. Inhibition is preferred to overcome this unfavorable overexpression.
A combination screening strategy was used to find a small molecule with the potential to be an inhibitor of miRNA production. This type of screening strategy was a hybrid of target- based screening and cell-based screening. The goal of this combination was to overcome the limitations of target-based screening, which did not take into account the physiological aspects of the living organism, and also cell-based screening, that considered as mechanism- agnostic screening. The combination of both types of screening would increase the relevance of screening results to physiological conditions and make it easier to track the mechanism of action behind the inhibition.
Uterine corpus endometrial carcinoma (UCEC) was chosen to apply those screening concepts due to the lack of alternative drugs available to cure this kind of cancer. Several UCEC-associated miRNAs reported by Favier A., et al. then were used to demonstrate the inhibition of small molecules to the production of UCEC-associated miRNAs (miR-182, miR-31, miR-30d). Nakatani group, on the other hand, developed small molecules with nucleobase recognition sites, such as guanine recognition by N-Acyl-2-amino-7-methyl-1,8-naphthyridine, adenine recognition by 7-methyl-2-oxo-1,8-naphthyridine (azaquinolone), and cytosine recognition by protonated 2-amino-7-methyl-1,8-naphthyridine. These small molecules are designed to specifically bind the complementary nucleobase on the nucleic acids. If those small molecules can bind to miRNA precursors, they may inhibit oncogenic miRNA production. The first chapter of this dissertation described the framework and background of the research. In chapter 2, the use of Real-time PCR (qPCR) to determine the kinetical properties of an in-vitro Dicer reaction was investigated. However, due to a number of challenges, the results of this experiment did not meet the expected results.
In chapter 3, We demonstrated a multistep qPCR-based screening of an in-house chemical library that targets UCEC-associated miRNA production using the screening strategy that combined the target-based screening and cell-based screening assay. In-vitro Dicer-mediated processing of pre-miR-182, pre-miR-31, and pre-miR-30d was used for the initial screening. The first screening yielded 48 different compounds with significant inhibition effects on miRNA production. The inhibitory effect of the identified compounds on the biogenesis of the miRNA targets on the cells was then investigated. We discovered eight hit compounds with potential inhibitory effects on pre-miR-182 and pre-miR-31 processing in vitro and in cells. Furthermore, the interaction of eight compounds with pre-miRNAs was studied using the Surface Plasmon Resonance (SPR) assay and gel analysis. Among eight hit compounds, only 2 compounds showed favorable binding to pre-miRNAs. This suggested two possible ways of inhibition may occur, first, the compounds bind to the pre-miRNA and interfere with the Dicer-mediated cleavage processing, or the compounds directly interact with Dicer and affected the cleavage processing.
