Identification Of Small-molecule MiRNA Modifiers And The Investigation On Their Mechanism Of Action

MicroRNAs (miRNAs) are a class of endogenous non-coding regulatory small RNAs, which regulate gene expression at post-transcriptional level by binding to the3’ untranslated regions (3’UTR) of specific mRNA targets to induce mRNA degradation or to repress mRNA transcription. The wide regulatory functions of miRNAs on gene expressions make them become attractive biomarkers and potential therapeutic targets for various diseases. Small molecules which can regulate the expression or function of miRNAs can act as molecular probe to elucidate miRNA-involved regulatory network inside cells. In this dissertation, the work on screening of small molecules that can regulate endogenous miRNAs was summarized and investigation on the regulatory mechanism of the active molecules on endogenous miRNAs was also included. The main work is described in the following four parts:1. Development of cellular evaluation system for screening of small molecules with regulatory activities on endogenous miRNAs. In this part, we constructed two kinds of reporter gene system, the Luciferase and EGFP C1reporter gene, in which the cellular specific miRNA affected by small molecule could be read out through bioluminescence signal or fluorescence signal. In the Luciferase reporter gene screening system, the corresponding miRNA complementary sequences was inserted into Luciferase3’UTR. In normal condition, the endogenous miRNA can bind with the miRNA target sequence, and the expression of Luciferase will be suppressed; upon incubation of compounds with the cells, the expression or function of the endogenous miRNA may be regulated, and the regulatory effect of small molecule on miRNA and its relative regulatory strength can be preliminary judged by the change of bioluminescence signal. The screening system of EGFP C1reporter gene is principally identical to Luciferase reporter gene screening system, but the fluorescence change after incubation with molecule need to be detected by flow cytometry, so it has some limitation on high throughput screening of active molecules.2. Evaluation on the regulatory activities of small molecules in various chemical blocks. Base on the screening system of Luciferase reporter gene in the first part, the activity of small molecules from our chemical blocks which synthesized from photoreaction and other synthetic methods was evaluated. Small molecules with negligible cytotoxicity at concentration of10μM were first selected and then the small molecules activities on cancer-related miR-21in HeLa cells, muscle specific miR-1and miR-133a in C2C12cells, and liver specific miR-122in HepG2cells were tested. This part of work provided some general information on the possibility of small molecules with different framework to act as speicifc or universal modifiers of miRNAs.3. The identification of universal activator of miRNAs and its mechanism of action. An active compound2a-4was identified from the photoreaction products of naphthalene-1,4-dione with diphenyl acetylene, which exhibited activation activity on miR-21, miR-1, miR-122and other types of miRNAs. The small molecule was considered as universal activator of miRNAs. Exploration on its mechanism of action revealed that the small-molecule activator up-regulated the expression level of mature miRNAs inside cells but down-regulated pre-miRNAs level significantly. The active molecule thus promote the processing of pre-miRNAs into mature miRNAs. Further investigation on the important proteins involved in the process of miRNA maturation, including AGO2and TRBP, revealed that the expression of TRBP in cells treated with the molecule was up-regulated. Therefore the small-molecule activator might promote the miRNA processing through enhancing the expression of TRBP. But the direct target of the active molecule and the complete regulatory pathway remained to be elucidated.4. Attempts to construct molecular probe for the exploration on the universal activation pathway. Based on the identified universal activator2a-4as described in the third part, further exploration on the activities of its analogues obtained via similar photoreactions of naphthalene-1,4-dione with various substituted phenyl acetylenes was conducted in this part. The objective on this part of part was to find the practical way to build molecular probe bearing bio-orthogonal group which remained to be universal activator of miRNAs like2a-4to elucidate the direct target and regulatory pathway of2a-4inside cells. Various analogues of2a-4with the common benzoanthracen-4-one framework were obtained by photocycloaddition reaction of a naphthalene-1,4-dione with various substituted phenyl acetylenes. Evaluation on the cytotoxicity and activity of the resulted analogues on miRNAs indicated that most of the analogues obtained in this part showed significant cytotoxicity to cells at the concentration of10μM. Lowering the concentration of compound at which no obvious cytotoxicity was observed resulted in significantly decreased activity on the activation of endogenous miRNAs. Other practicle structural modification on alternative positions of2a-4remains to be explored to construct useful molecular probe and the work is now undergoing in our group.