Postdoctoral Researcher, University of Michigan
Engineering synthetic toehold switch to detect microRNA expression in cells
microRNAs (miRNA) are small, non-coding RNA molecules with the length of 21-23 nucleotides that have been demonstrated to play critical roles in different biological processes including development, proliferation, and apoptosis by mediating translational repression through targeting messenger RNA. Increasing evidence have shown miRNA plays an important role in human diseases including cancer and immune disorders. Furthermore, current studies indicate that miRNAs can function either as oncogenes or tumor suppressors which play a key role in the pathogenesis of cancer. Thus, there is a quest to understand miRNAs regulated signaling pathway and to effectively target oncogenic miRNAs or tumor-suppressive miRNAs for anticancer therapy. Current miRNA-based cancer therapy includes delivery of miRNA mimics and anti-miRNAs to restore miRNAs expression levels. However, these conventional approaches are based on the hypothesis that the miRNA expression levels are known, overexpression or expressed at low levels. Thus, sensing and programming the internal states of living cells based on miRNA expression levels represents a major and formidable challenge in realizing the therapeutic potential of engineered cells. Recent advances in genome editing tools have truly revolutionized biomedical research. Yet, a strategy that integrates a sensing and processing mechanism is still lacking. In the context of cancer research, a number of scenarios could benefit from the platform that we propose to develop. The objective of this proposal is to develop a novel synthetic biology platform for sensing and programming cancer cells for the development of microRNA-based anticancer therapeutics.
Abstract: Angiogenesis is a complex cellular process involving highly orchestrated invasion and organization of endothelial cells (ECs) in a three-dimensional (3D) environment. Recent evidence indicates that Notch signaling is critically involved in regulating specialized functions and distinct fates of ECs in newly formed vasculatures during angiogenesis. Here, we demonstrated, for the first time, the application of a microengineered biomimetic system to quantitatively investigate the role of Notch signaling in regulating early angiogenic sprouting and vasculature formation of ECs in a 3D extracellular matrix. Morphological features of angiogenesis including invasion distance, invasion area, and tip cell number were quantified and compared under pharmacological perturbations of Notch signaling. In addition, influences of Notch signaling on EC proliferation in angiogenic vasculatures and directional invasion of tip cells were also investigated. Moreover, leveraging a novel nanobiosensor system, mRNA expression of Dll4, a Notch ligand, was monitored in invading tip cells using live cell imaging during the dynamic angiogenic process. Our data showed that inhibition of Notch signaling resulted in hyper-sprouting endothelial structures, while activation of Notch signaling led to opposite effects. Our results also supported the role of Notch signaling in regulating EC proliferation and dynamic invasion of tip cells during angiogenesis.
Pub.: 05 May '17, Pinned: 21 Aug '17
Abstract: The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.
Pub.: 22 Mar '14, Pinned: 21 Aug '17
Abstract: The formation of microvascular networks plays essential roles in regenerative medicine and tissue engineering. Nevertheless, the self-organization mechanisms underlying the dynamic morphogenic process are poorly understood due to a paucity of effective tools for mapping the spatiotemporal dynamics of single cell behaviors. By establishing a single cell nanobiosensor along with live cell imaging, we perform dynamic single cell analysis of the morphology, displacement, and gene expression during microvascular self-organization. Dynamic single cell analysis reveals that endothelial cells self-organize into subpopulations with specialized phenotypes to form microvascular networks and identifies the involvement of Notch1-Dll4 signaling in regulating the cell subpopulations. The cell phenotype correlates with the initial Dll4 mRNA expression level and each subpopulation displays a unique dynamic Dll4 mRNA expression profile. Pharmacological perturbations and RNA interference of Notch1-Dll4 signaling modulate the cell subpopulations and modify the morphology of the microvascular network. Taken together, a nanobiosensor enables a dynamic single cell analysis approach underscoring the importance of Notch1-Dll4 signaling in microvascular self-organization.
Pub.: 23 Aug '16, Pinned: 21 Aug '17
Abstract: At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct 'leader' phenotype with characteristic morphology and motility. However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here we use single-cell gene expression analysis and computational modelling to show that the leader cell identity is dynamically regulated by Dll4 signalling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.
Pub.: 15 Mar '15, Pinned: 21 Aug '17
Abstract: A gold nanorod-locked nucleic acid nano-biosensor for dynamic single-cell gene expression analysis in living cells and tissues is developed. The nanoparticle facilitates endocytic delivery and dynamic monitoring of the gene expression in human umbilical cord endothelial cells, mouse skin tissues, mouse retina tissues, and mouse cornea tissues at the single-cell level.
Pub.: 01 Sep '15, Pinned: 21 Aug '17
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