Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.     

Regulators of cholinergic signaling in disorders of the central nervous system

Cholinergic signaling is crucial in cognitive processes, and degenerating cholinergic projections are a pathological hallmark in dementia. Use of cholinesterase inhibitors is currently the main treatment option to alleviate symptoms of Alzheimer's disease and has been postulated as a therapeutic strategy in acute brain damage (stroke and traumatic brain injury). However, the benefits of this treatment are still not clear. Importantly, cholinergic receptors are expressed both by neurons and by astrocytes and microglia, and binding of acetylcholine to the α7 nicotinic receptor in glial cells results in anti-inflammatory response. Similarly, the brain fine-tunes the peripheral immune response over the cholinergic anti-inflammatory axis. All of these processes are of importance for the outcome of acute and chronic neurological disease. Here, we summarize the main findings about the role of cholinergic signaling in brain disorders and provide insights into the complexity of molecular regulators of cholinergic responses, such as microRNAs and transfer RNA fragments, both of which may fine-tune the orchestra of cholinergic mRNAs. The available data suggest that these small noncoding RNA regulators may include promising biomarkers for predicting disease course and assessing treatment responses and might also serve as drug targets to attenuate signaling cascades during overwhelming inflammation and to ameliorate regenerative capacities of neuroinflammation.

     

Immunoamphisomes in dendritic cells amplify TLR signaling and enhance exogenous antigen presentation on MHC-II

Autophagy, a specialized lysosomal degradation pathway, has proven to be a potent cell-autonomous defense mechanism against a range of intracellular microbes. In addition, autophagy emerged recently as a critical regulator of innate and adaptive immune responses. Links between autophagy and innate immunity are being progressively unveiled. For instance, several TLR (Toll-Like Receptor) agonists upregulate autophagy flux in immune cell types such as DC (dendritic cells) or macrophages. Conversely, and perhaps surprisingly, is the observation that TLR7-mediated responses might depend on autophagy in plasmacytoid DC, thus suggesting a more complex link between TLR-dependent responses and autophagy. Recently, the demonstration that NOD2 increases autophagy suggests that innate immune responses initiated via a broad range of pathogen recognition receptors can regulate autophagy. In addition to its involvement in innate immune responses, autophagy regulates adaptive immune responses via both MHC class I and class II molecules depending on the cellular context and the nature of the antigen.     

Inhibition of Cell Proliferation and Migration by miR-509-3p That Targets CDK2, Rac1, and PIK3C2A

CDK2 is a key regulator of cell cycle progression. In this study, we screened for miRNAs targeting CDK2 using a luciferase-3′-untranslated region reporter assay. Among 11 hit miRNAs, miR-509-3p reduced CDK2 protein levels and significantly inhibited cancer cell growth. Microarray, Western blotting, and luciferase reporter analyses revealed additional targets of miR-509-3p, including Rac1 and PIK3C2A. Overexpression of miR-509-3p induced G1 cell-cycle arrest and inhibited colony formation and migration. RNAi experiments indicated that the growth-inhibitory effects of miR-509-3p may occur through down-regulation of CDK2, Rac1, and PIK3C2A. Targeting of multiple growth regulatory genes by miR-509-3p may contribute to effective anti-cancer therapy.     

Let-7e enhances the radiosensitivity of colorectal cancer cells by directly targeting insulin-like growth factor 1 receptor

Abnormal expression of various microRNAs (miRNAs), as regulators of biological signaling pathways, has a strong association with cancer resistance to chemotherapy and radiotherapy. The let-7 family of miRNAs as tumor suppressors have shown to be downregulated in different types of human malignancies including colorectal cancer (CRC). However, the biological function of let-7 members in the processes of resistance to radiation in CRC has not yet been completely elucidated. Insulin-like growth factor 1 receptor (IGF-1R) signaling pathway is amplified in CRC and leads to its progression, development, and also radiation resistance. So, it seems like an attractive target for anticancer therapy. In this study, by using bioinformatics analysis, it has been revealed that IGF-1R is a direct target of the let-7e member. Consistent with this, we identified that increased levels of let-7e in CRC cells reduced IGF-1R protein level and subsequently its downstream signaling pathways, which resulted in the G1 cell cycle arrest and a significant reduction in the proliferation, survival and also resistance to radiation of CRC cells. Altogether, these results suggested that let-7e by targeting the IGF-1R signaling pathway might serve as therapeutics in anticancer therapy.     

H3K4 di-methylation governs smooth muscle lineage identity and promotes vascular homeostasis by restraining plasticity

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miR-15b induced by platelet-derived growth factor signaling is required for vascular smooth muscle cell proliferation

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA-15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs. [BMB Reports 2013; 46(11): 550-554]