The Screening Of MiR-21Small Molecule Inhibitor And Experimental Studying On Tumor Targeting Therapy

MicroRNAs (miRNAs or miRs) have attracted significant attention as a new class of envolutionary conserved noncoding-RNAs engaged in the regulation of gene expression. miRNAs are short19-25nt stretches of RNA that bind to the target mRNAs and suppress their translation or initiate degradation. There are more than1000miRNAs identified in human genome currently. The miRNAs expression sequencing indicated that the expression signatures may function as specific tumor markers, which are associated with cancer staging, diagnosis, prognosis and response to treatment. As a result, explore the representative and differentially expressed miRNAs, establishing their cancer-associated functions and developing drugs to target their activities, is promising therapeutic strategies against cancer.miR-21was one of the first miRNAs detected in the human genome and it has been validated to be overexpressed in many different types of human cancers, such as glioblastomas, gastric tumors, breast cancers, colon cancers and lung cancers. Meanwhile, miR-21targeted multiple mRNAs to modulate many biologic pathways related to cancer initiation, progression, metastasis, and therapy resistance. Currently, non-small molecular nucleotide agents against miRNAs were not ideal for the clinical application due to the challenges involved in the delivery of these agents, and their pharmacodynamics and pharmacokinetics properties. Therefore, the development of small-molecule drugs targeting specific miRNAs and modulating their activities would be a promising approach.In the first part of our study, we proposed a computer aided drug design for small molecules targeting miR-21based on the3D structure of the Dicer binding site on pre-miR-21and achieved the candidate compounds after high-throughput molecular docking for pre-miR-21against the NCI/diversity compounds. AC1MMYR2was identified as a promising miR-21inhibitor after inhibitory efficacy assay in vitro. AC1MMYR2decreased the miR-21expressions in a dose-and time-dependent manner, but induced the PTEN, PDCD4and RECK expressions. Moreover, only miR-21exrpression were reduced after AC1MMYR26h treatment, whereas the expression levels miR-1280, miR-218and miR-200a/b were induced after24h treatment. In addition, in comparison with miR-21low-expressed tumor cells, AC1MMYR2was more effective in miR-21high-expression cells or in other words, to some extent, AC1MMYR2selectively targets miR-21-high tumors.In the second part of our study, besides the reduction of mature miR-21expression induced by AC1MMYR2treatment, we found pri-miR-21expressions and STAT3transcriptional activity were also reduced, whereas the levels of pre-miR-21were increased. Further studies indicated that AC1MMYR2blocked Dicer processing at the binding site of pre-miR-21to repress the mature miR-21generation. Meanwhile, our previous study reported there was binding site of STAT3on the upstream of miR-21promoter thereby promoted miR-21expression. So we speculated that the underlying mechanism of reduced pri-miR-21expressions partial due to the AC1MMYR2-STAT3-pri-miR-21feedback loop. To analysis whether the point mutations influenced AC1MMYR2action, we created the3D structure of pre-miR-21hairpin loop with such mutations, we confirmed that point mutations at Dicer binding site might attenuate AC1MMYR2binding affinity and thus weaken its activity. Therefore, AC1MMYR2probable specifically blocked the Dicer binding site on pre-miR-21. In addition, we also found AC1MMYR2functions are dependent on Dicer expression. The compound could not effectively repress mature miR-21generation when Dicer levels were low.In the third part of our study, we mainly investigated the impact of AC1MMYR2on biological characteristic of five epithelial cancer cell lines. AC1MMYR2reduced the tumor cell colony formation rate and blocked the cell cycle in the GO-Gl phase to inhibit the cell proliferation. Meanwhile the apoptosis rate and Caspase3/7activity was increased, whereas the migration and invasion was inhibited after AC1MMYR2treatment. Furthermore, AC1MMYR2treatment induced the expression of E-cadherin, whereas the mesenchymal markers of N-cadherin,(3-catenin, ZEB1, ZEB2and MMP9expressions were reduced, which indicated the EMT was reversed in a sense. In addition, KEGG pathway analysis was conducted on the534genes whose expression was significantly altered in response to6-hour treatment with AC1MMYR2in U87glioma cells. There were14different pathways corresponding to the target genes, of which "pathways in cancer" and "TGF-(3pathway" were top two most significant. These indicated AC1MMYR2efficacy may be associated with cancer and EMT process.In the last part, U87glioma intracranial model and MCF-7and MDA-MB-231breast cancer orthotopic models were preformed to further analysis the AC1MMYR2antitumor efficacy. After AC1MMYR2treatment, the tumor growth was significantly inhibited. Meanwhile, AC1MMYR2treatment improved the prognosis of U87intracranial model mice and reduced the MDA-MB-231cells lung metastasis. H&E staining of multiple tissues showed no discernible toxicity of AC1MMYR2, including in the liver and kidney. In addition, we also detected BASI (NSC-211332), a non-specific miR-21inhibitor, antitumor efficacy in U87intracranial glioma model. We found that the compound inhibited the tumor growth and improved the mouse prognosis via alteration of several β-catenin associated miRNAs (miR-21, miR-23b, miR-200a, miR-181d), which ultimately suppressed the Wnt/β-catenin signaling pathway.Based on these studies, we propose a novel high-throughput screening method for small molecules targeting miRNAs and identify a potent and specific miR-21inhibitor, AC1MMYR2, which shows remarkable antitumor efficacy not only through an induction of common functional targets (PTEN, PDCD4, and RECK) but also the reversion of EMT through upregulation of miR-200a/b and miR-181d; Taken together, our results showed the robust antitumor efficacy of AC1MMYR2without any observable toxicity to normal tissues, which highlighted its promising use clinically. AC1MMYR2compound derivatives might have potential as anticancer agents in epithelial cancers in the future.