Intracellular microRNA Research Articles

Synthesis of fluorescent dye-doped silica nanoparticles for target-cell-specific delivery and intracellular microRNA imaging.

MicroRNA (miRNA) is found to be up-regulated in many kinds of cancer and therefore is classified as an oncomiR. Herein, we design a multifunctional fluorescent nanoprobe (FSiNP-AS/MB) with the AS1411 aptamer and a molecular beacon (MB) co-immobilized on the surface of the fluorescent dye-doped silica nanoparticles (FSiNPs) for target-cell-specific delivery and intracellular miRNA imaging. The FSiNPs were prepared by a facile reverse microemulsion method from tetraethoxysilane and silane derivatized coumarin that was previously synthesized by click chemistry. The as-prepared FSiNPs possess uniform size distribution, good optical stability and biocompatibility. In addition, there is a remarkable affinity interaction between the AS1411 aptamer and the nucleolin protein on the cancer cell surface. Thus, a target-cell-specific delivery system by the FSiNP-AS/MB is proposed for effectively transferring a MB into the cancer cells to recognize the target miRNA. Using miRNA-21 in MCF-7 cells (a human breast cancer cell line) as a model, the proposed multifunctional nanosystems not only allow target-cell-specific delivery with the binding affinity of AS1411, but also can track simultaneously the transfected cells and detect intracellular miRNA in situ. The proposed multifunctional nanosystems are a promising platform for a highly sensitive luminescent nonviral vector in biomedical and clinical research.

In situ amplification of intracellular microRNA with MNAzyme nanodevices for multiplexed imaging, logic operation, and controlled drug release.

MicroRNAs (miRNAs), as key regulators in gene expression networks, have participated in many biological processes, including cancer initiation, progression, and metastasis, indicative of potential diagnostic biomarkers and therapeutic targets. To tackle the low abundance of miRNAs in a single cell, we have developed programmable nanodevices with MNAzymes to realize stringent recognition and in situ amplification of intracellular miRNAs for multiplexed detection and controlled drug release. As a proof of concept, miR-21 and miR-145, respectively up- and down-expressed in most tumor tissues, were selected as endogenous cancer indicators and therapy triggers to test the efficacy of the photothermal nanodevices. The sequence programmability and specificity of MNAzyme motifs enabled the fluorescent turn-on probes not only to sensitively profile the distributions of miR-21/miR-145 in cell lysates of HeLa, HL-60, and NIH 3T3 (9632/0, 14147/0, 2047/421 copies per cell, respectively) but also to visualize trace amounts of miRNAs in a single cell, allowing logic operation for graded cancer risk assessment and dynamic monitoring of therapy response by confocal microscopy and flow cytometry. Furthermore, through general molecular design, the MNAzyme motifs could serve as three-dimensional gatekeepers to lock the doxorubicin inside the nanocarriers. The drug nanocarriers were exclusively internalized into the target tumor cells via aptamer-guided recognition and reopened by the endogenous miRNAs, where the drug release rates could be spatial-temporally controlled by the modulation of miRNA expression. Integrated with miRNA profiling techniques, the designed nanodevices can provide general strategy for disease diagnosis, prognosis, and combination treatment with chemotherapy and gene therapy.

Magnetic resonance beacon to detect intracellular microRNA during neurogenesis

Magnetic resonance imaging (MRI) offers great spatial resolution for viewing deep tissues and anatomy. We developed a self-assembling signal-on magnetic fluorescence nanoparticle to visualize intracellular microRNAs (miRNAs or miRs) during neurogenesis using MRI. The self-assembling nanoparticle (miR124a MR beacon) was aggregated by the incubation of three different oligonucleotides: a 3′ adaptor, a 5′ adaptor, and a linker containing miR124a-binding sequences. The T2-weighted magnetic resonance (MR) signal of the self-assembled nanoparticle was quenched when miR124a was absent from test tubes or was minimally expressed in cells and tissues. When miR124a was present in test tubes or highly expressed in vitro and in vivo during P19 cell neurogenesis, it hybridized with the miR124a MR beacon, causing the linker to detach, resulting in increased signal-on MRI intensity. This MR beacon can be used as a new imaging probe to monitor the miRNA-mediated regulation of cellular processes.

Integrated Computational Model of Intracellular Signaling and microRNA Regulation Predicts the Network Balances and Timing Constraints Critical to the Hepatic Stellate Cell Activation Process

Activation and deactivation of hepatic stellate cells (HSCs) is an important mechanism contributing to both healthy liver function and development of liver diseases, which relies on the interplay between numerous signaling pathways. There is accumulating evidence for the regulatory role of microRNAs that are downstream from these pathways in HSC activation. However, the relative contribution of these pathways and interacting microRNA regulators to the activation process is unknown. We pursued a computational modeling approach to explore the timing and regulatory balances that are critical to HSC activation and quiescence. We developed an integrated model incorporating three signaling pathways with crosstalk (NF-κB, STAT3 and TGF-β) and two microRNAs (miR-146a, miR-21) that are differentially regulated by these pathways. Simulations demonstrated that TGF-β-mediated regulation of microRNAs is critical to drive the HSC phenotypic switch from quiescence (miR-146ahigh miR-21low) to an activated state (miR-146alow miR-21high). We found that the relative timing between peak NF-κB and STAT3 activation plays a key role driving the initial dynamics of miR-146a. We observed re-quiescence from the activated HSC state upon termination of cytokine stimuli. Our integrated model of signaling and microRNA regulation provides a new computational platform for investigating the mechanisms driving HSC molecular state phenotypes in normal and pathological liver physiology.

Simultaneous sensing of intracellular microRNAs with a multi-functionalized carbon nitride nanosheet probe.

A multi-functionalized CNNS probe was designed by loading different dye-ssDNAs and folate on CNNSs, which was used for in situ fluorescence imaging and sensing of intracellular multiple microRNAs.

The role of microRNAs in cellular senescence and age-related conditions of cartilage and bone

Background and purpose — We reviewed the current state of research on microRNAs in age-related diseases in cartilage and bone. Methods — PubMed searches were conducted using separate terms to retrieve articles on (1) the role of microRNAs on aging and tissue degeneration, (2) specific microRNAs that influence cellular and organism senescence, (3) microRNAs in age-related musculoskeletal conditions, and (4) the diagnostic and therapeutic potential of microRNAs in age-related musculoskeletal conditions. Results — An increasing number of studies have identified microRNAs associated with cellular aging and tissue degeneration. Specifically in regard to frailty, microRNAs have been found to influence the onset and course of age-related musculoskeletal conditions such as osteoporosis, osteoarthritis, and posttraumatic arthritis. Both intracellular and extracellular microRNAs may be suitable to function as diagnostic biomarkers. In particular Interpretation — The research data currently available suggest that microRNAs play an important role in orchestrating age-related processes and conditions of the musculoskeletal system. Further research may help to improve our understanding of the complexity of these processes at the cellular and extracellular level. The option to develop microRNA biomarkers and novel therapeutic agents for the degenerating diseases of bone and cartilage appears to be promising.

Potential molecular and cellular mechanism of psychotropic drugs.

Psychiatric disorders are among the most debilitating of all medical illnesses. Whilst there are drugs that can be used to treat these disorders, they give sub-optimal recovery in many people and a significant number of individuals do not respond to any treatments and remain treatment resistant. Surprisingly, the mechanism by which psychotropic drugs cause their therapeutic benefits remain unknown but likely involves the underlying molecular pathways affected by the drugs. Hence, in this review, we have focused on recent findings on the molecular mechanism affected by antipsychotic, mood stabilizing and antidepressant drugs at the levels of epigenetics, intracellular signalling cascades and microRNAs. We posit that understanding these important interactions will result in a better understanding of how these drugs act which in turn may aid in considering how to develop drugs with better efficacy or increased therapeutic reach.

Regulation of microRNA-155 in endothelial inflammation by targeting nuclear factor (NF)-κB P65.

Increasing evidences have illuminated the fundamental role of inflammation in mediating all stages of atherosclerosis. miR-155, a typical multi-functional miRNA, has recently emerged as a novel component of inflammatory signal transduction in the pathogenesis of atherosclerosis. However, little is known about whether endothelial highly expressed miR-155 can regulate endothelial inflammation-related transcription factors and the predicted role of miR-155 as a negative feedback regulator in endothelial inflammation involved in atherosclerosis. Bioinformatics analysis showed that RELA (nuclear factor-κB p65) is a potential target gene of miR-155 and this was confirmed by a luciferase reporter assay. Our results show that microRNA-155 mediate endothelial inflammation and decrease NFкB p65 and adhesion molecule expression in TNFα-stimulated endothelial cells. Transfection with miR-155 significantly inhibited TNFα-induced monocyte adhesion to endothelium. Inhibition of miR-155 enhanced p65 level and endothelial inflammatory response which was counteracted through the depletion of P65 by Si-P65. On the other hand, knockdown of eNOS, another target of miR-155, while transfecting with miR-155 inhibitor resulted in more significant inflammatory response. miR-155 is highly expressed in TNFα treated HUVECs, deprived of endogenous p65 could reverse TNFα-induced upregulation of miR-155. Thus, TNFα induced miR-155 may serve as a negative feedback regulator in endothelial inflammation involved in atherosclerosis by targeting nuclear transcription factor P65. These results provide a rationale for intervention of intracellular microRNA as possible anti-atherosclerotic targets.

Extracellular MicroRNAs Activate Nociceptor Neurons to Elicit Pain via TLR7 and TRPA1

Intracellular microRNAs (miRNAs) are key regulators of gene expression. The role of extracellular miRNAs in neuronal activation and sensory behaviors are unknown. Here we report an unconventional role of extracellular miRNAs for rapid excitation of nociceptor neurons via toll-like receptor-7 (TLR7) and its coupling to TRPA1 ion channel. miRNA-let-7b induces rapid inward currents and action potentials in dorsal root ganglion (DRG) neurons. These responses require the GUUGUGU motif, only occur inneurons coexpressing TLR7 and TRPA1, and are abolished in mice lacking Tlr7 or Trpa1. Furthermore, let-7b induces TLR7/TRPA1-dependent single-channel activities in DRG neurons and HEK293 cells overexpressing TLR7/TRPA1. Intraplantar injection of let-7b elicits rapid spontaneous pain via TLR7 and TRPA1. Finally, let-7b can be released from DRG neurons by neuronal activation, and let-7b inhibitor reduces formalin-induced TRPA1 currents and spontaneous pain. Thus, secreted extracellular miRNAs may serve as novel pain mediators via activating TLR7/TRPA1 in nociceptor neurons.

Contribution of microRNA 24–3p and Erk1/2 to interleukin‐6‐mediated plasma cell survival

Plasma cells can survive for long periods and continuously secrete protective antibodies, but plasma cell production of autoantibodies or transformation to tumor cells is detrimental. Plasma cell survival depends on exogenous factors from the surrounding microenvironment, and largely unknown intracellular mediators that regulate cell homeostasis. Here we investigated the contribution of the microRNA 24-3p (miR-24-3p) to the survival of human plasma cells under the influence of IL-6 and SDF-1α (stromal cell derived factor 1), both of which are bone marrow survival niche mediators. Deep sequencing revealed a strong expression of miR-24-3p in primary B cells, plasma blasts, plasma cells, and in plasmacytoma cells. In vitro studies using primary cells and the plasmacytoma cell line RPMI-8226 revealed that (i) expression of miR-24-3p mediates plasma cell survival, (ii) miR-24-3p is upregulated by IL-6 and SDF-1α, (iii) IL-6 mediates cell survival under ER stress conditions via miR-24-3p expression, and (iv) IL-6-induced miR-24-3p expression depends on the activity of the MAP kinase Erk1/2. These results suggest a direct connection between an external survival signal and an intracellular microRNA in regulating plasma cell survival. miR-24-3p could therefore be a promising target for new therapeutic strategies for autoimmune and allergic diseases and for multiple myeloma.

Fluctuation of Global Gene Expression by Endogenous miRNA Response to the Introduction of an Exogenous miRNA

Most of the intracellular endogenous microRNAs (endo-miRNAs) are considered to be saturated in Argonaute (Ago) proteins in the RNA-induced silencing complexes (RISCs). When exogenous miRNAs (exo-miRNAs) are introduced into cells, endo-miRNAs in the RISC may be replaced with exo-miRNAs or exo-miRNAs, and endo-miRNAs might also compete for the position in the newly synthesized RISC with each other. This would lead to the fluctuation of global gene expression not only by repression of exo-miRNA target gene expression, but also by the increase of the endo-miRNA target gene expression. In the present study, we quantified the changes in the expression levels of target genes of exo-miRNA and endo-miRNA in the cells transfected with fifteen different exo-miRNAs by microarray experiments. Different exo-miRNAs increased ratios of expression levels of target genes of a given endo-miRNA to different extents, suggesting that the replacement efficiencies might differ according to the exo-miRNA types. However, the increased ratios in the expression levels of each endo-miRNA target genes by the transfection of any particular exo-miRNA were mostly equivalent, suggesting that the endo-miRNAs present in the RISC might be replaced with excessive exo-miRNAs at similar levels, probably because they exist in single-stranded forms in the RISC.

Synthetic microRNA-mediated downregulation of Nogo-A in transgenic rats reveals its role as regulator of synaptic plasticity and cognitive function

We have generated a transgenic rat model using RNAi and used it to study the role of the membrane protein Nogo-A in synaptic plasticity and cognition. The membrane protein Nogo-A is expressed in CNS oligodendrocytes and subpopulations of neurons, and it is known to suppress neurite growth and regeneration. The constitutively expressed polymerase II-driven transgene was composed of a microRNA-targeting Nogo-A placed into an intron preceding the coding sequence for EGFP, thus quantitatively labeling cells according to intracellular microRNA expression. The transgenic microRNA in vivo efficiently reduced the concentration of Nogo-A mRNA and protein preferentially in neurons. The resulting significant increase in long-term potentiation in both hippocampus and motor cortex indicates a repressor function of Nogo-A in synaptic plasticity. The transgenic rats exhibited prominent schizophrenia-like behavioral phenotypes, such as perseveration, disrupted prepulse inhibition, and strong withdrawal from social interactions. This fast and efficient microRNA-mediated knockdown provides a way to silence gene expression in vivo in transgenic rats and shows a role of Nogo-A in regulating higher cognitive brain functions.

Mapping of the 3′ end for artificial human microRNAs

The 3' end was exactly mapped for has-mir-30a-like artificial human microRNAs that specifically recognize the mRNA of the aml1/eto fusion oncogene. The results indicated that the intracellular microRNA pool was heterogeneous in linear size relative to the 3' end, which is necessary to consider in designing and using artificial microRNAs specific for mRNAs of other genes.

Target‐Cell‐Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO2 Nanoprobe

MicroRNAs (miRNAs) are a class of short, endogenous, noncoding regulatory RNAs (approximately 18–25 nucleotides), encoded in the genomes of plants, animals, and viruses. They partially complement the 3’ untranslated region of target mRNAs, causing mRNA cleavage or inhibiting protein synthesis within the Dicer/Argonaute complex. MiRNAs play key regulatory roles in a diverse range of biological processes, including cell development, differentiation, metabolism, and apoptosis. In particular, distinct miRNA expression patterns are associated with various cancer phenotypes. Therefore, miRNAs are also an emerging class of useful diagnostic and prognostic markers. However, miRNA analysis is challenging owing to the unique characteristics of miRNAs, such as their small size, sequence similarity among family members, low abundance, susceptibility to degradation, and the technical impediments in delivering miRNAs across the plasma membrane of cells. An improved delivery strategy is thus needed to successfully monitor intracellular miRNA levels in situ. Generally, transmission of oligonucleotides for gene therapy falls into two broad categories, viral vectors or nonviral carriers. Viral vectors exhibit high transfection efficiency but also some fundamental problems, such as immunogenicity and toxicity. These problems limit their broad application. Currently, the nonviral vectors, such as liposomes, cationic polymers, dendrimers, polypeptides, and nanomaterials have attracted significant interest, owing to their good biocompatibility and potential for large-scale production, in contrast to viral vectors. However, many of these carrier systems do not inhibit genes in a cell-specific manner and cannot be used to monitor their intracellular delivery or therapeutic response. An excellent nonviral vector should efficiently facilitate cell-specific gene-probe (a single-stranded oligonucleotide designed to recognize a single target-nucleotide sequence) uptake and gene-probe endosomal escape for intracellular delivery. Fluorescent nanoparticles are highly attractive materials for gene-probe delivery because of their unique properties, including uniform size, superior imaging characteristics, and facile surface modification. The rational functionalization of these nanomaterials with target-specific moieties, such as antibodies, aptamers, and other molecules, to recognize receptors on the cell surface has led to versatile theragnostic nanosystems. These multifunctional nanosystems not only allow efficient delivery of gene probes to target cells with fewer side effects, but also allow the simultaneous monitoring of delivery and the therapeutic response of the targeted genes, using the advanced optical properties of the nanosystem. However, these nanosystems have not been used for in situ detection of intracellular miRNAs yet, because the detection strategy has not been optimized. In this study we design a novel multifunctional SnO2 nanoprobe (mf-SnO2), which contains a cell-targeting moity, as well as a conjugated gene probe to specifically recognize the target sequence, thus providing a detection strategy or inhibitor. Also, visualization of the delivery and intracellular response is possible through fluorescence of the SnO2. As shown in Scheme 1, cell-specific delivery is achieved by functionalizing SnO2 nanoparticles (SnO2NPs) with folic acid (FA), which targets cancer cells; a gene probe, in this case a molecular beacon (MB) to detect target miRNAs, is conjugated by a disulfide linkage, which is sensitive to pH values. Cleavage of the disulfide linkage between the gene probe and the nanoparticle enhances the efficiency of intracellular delivery. Using miRNA-21 in HeLa cells as a model, a method for in situ detection of intracellular miRNA by the multifunctional nanoprobe is reported. To verify the practicality of this approach, another nanoprobe was also designed, by substituting the MB with an anti-miR of miRNA-21, to down-regulate the expression of a target miRNA. The proposed method, with a MB as the recognition probe, can be used subsequently to monitor the change in miRNA levels from negligible cytotoxicity, and to monitor the ability of the multifunctional nanoprobe to [*] Dr. H. Dong, Prof. J. Lei, Prof. H. Ju, Z. Zhu State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University Nanjing 210093 (P.R. China) E-mail: hxju@nju.edu.cn

Target‐Cell‐Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO2 Nanoprobe

Target‐Cell‐Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO₂ Nanoprobe

Intracellular and Extracellular MicroRNAs in Breast Cancer

Successful treatment of breast cancer is enhanced by early detection and, if possible, subsequent patient-tailored therapy. Toward this goal, it is essential to identify and understand the most relevant panels of biomarkers, some of which may also have relevance as therapeutic targets. We critically reviewed published literature on microRNAs (miRNAs) as relevant to breast cancer. Since the initial recognition of the association of miRNAs with breast cancer in 2005, studies involving cell lines, in vivo models, and clinical specimens have implicated several functions for miRNAs, including suppressing oncogenesis and tumors, promoting or inhibiting metastasis, and increasing sensitivity or resistance to chemotherapy and targeted agents in breast cancer. For example, miR-21 is overexpressed in both male and female breast tumors compared with normal breast tissue and has been associated with advanced stage, lymph node positivity, and reduced survival time. miR-21 knock-down in cell-line models has been associated with increased sensitivity to topotecan and taxol in vitro and the limitation of lung metastasis in vivo. Furthermore, the discovery of extracellular miRNAs (including miR-21), existing either freely or in exosomes in the systemic circulation, has led to the possibility that such molecules may serve as biomarkers for ongoing patient monitoring. Although additional investigations are necessary to fully exploit the use of miRNAs in breast cancer, there is increasing evidence that miRNAs have potential not only to facilitate the determination of diagnosis and prognosis and the prediction of response to treatment, but also to act as therapeutic targets and replacement therapies.

Analysis of deep sequencing microRNA expression profile from human embryonic stem cells derived mesenchymal stem cells reveals possible role of let-7 microRNA family in downstream targeting of Hepatic Nuclear Factor 4 Alpha

BackgroundRecent literature has revealed that genetic exchange of microRNA between cells can be essential for cell-cell communication, tissue-specificity and developmental processes. In stem cells, as in other cells, this can be accomplished through microvesicles or exosome mediated transfer. However, molecular profiles and functions of microRNAs within the cells and in their exosomes are poorly studied. Next generation sequencing technologies could provide a broad-spectrum of microRNAs and their expression and identify possible microRNA targets. In this work, we performed deep sequencing of microRNAs to understand the profile and expression of the microRNAs in microvesicles and intracellular environment of human embryonic stem cells derived mesenchymal stem cells (hES-MSC).We outline a workflow pertaining to visualizing, statistical analysis and interpreting deep sequencing data of known intracellular and extracellular microRNAs from hES-MSC). We utilized these results of which directed our attention towards establishing hepatic nuclear factor 4 alpha (HNF4A) as a downstream target of let-7 family of microRNAs.ResultsIn our study, significant differences in expression profile of microRNAs were found in the intracellular and extracellular environment of hES-MSC. However, a high level of let-7 family of microRNAs is predominant in both intra- and extra- cellular samples of hES-MSC. Further results derived from visualization of our alignment data and network analysis showed that let-7 family microRNAs could affect the downstream target HNF4A, which is a known endodermal differentiation marker. The elevated presence of let-7 microRNA in both intracellular and extra cellular environment further suggests a possible intercellular signalling mechanism through microvesicles transfer. We suggest that let-7 family microRNAs might play a signalling role via such a mechanism amongst populations of stem cells in maintaining self renewal property by suppressing HNF4A expression. This is in line with recent paradigm where microRNAs regulate self-renewal and differentiation pathways of embryonic stem cells by forming an integral biological network with transcription factors.ConclusionIn summary, our study using a combination of alignment, statistical and network analysis tools to examine deep sequencing data of microRNAs in hES-MSC has led to a result that (i) identifies intracellular and exosome microRNA expression profiles of hES-MSCwith a possible mechanism of miRNA mediated intercellular regulation by these cells and (ii) placed HNF4A within the cross roads of regulation by the let-7 family of microRNAs.