Theragnosis by a miR-141-3p molecular beacon: simultaneous detection and sensitization of 5-fluorouracil resistant colorectal cancer cells through the activation of the TRIM13-associated apoptotic pathway.

We developed a molecular beacon targeting miR-141-3p, aberrantly increased in 5-fluorouracil-resistant colorectal cancer cells (R-CRCCs). It consists of a fluorophore-labeled oligonucleotide, antisense to miR-141-3p, and a quencher. It detected R-CRCCs and recovered the chemosensitivity of them to 5-fluorouracil by hybridization with miR-141-3p, which is applicable to cancer treatment.

An ultra-effective method of generating extramultipotent cells from human fibroblasts by ultrasound.

Multipotent cells have similar basic features of all stem cells but limitation in ability of self-renewal and differentiation compared with pluripotent cells. Here, we have developed an ultra effective, gene- and chemical-free method of generating extra multipotent (xpotent) cells which have differentiation potential more than limited cell types, by the mechanism of ultrasound-directed permeation of environmental transition-guided cellular reprogramming (Entr). Ultrasound stimulus generated a massive number of Entr-mediated xpotent (x/Entr) spheroids from human dermal fibroblasts (HDFs) 6 days after treatment. The emergence of x/Entr was first initiated by the introduction of human embryonic stem cell (ESC) environments into the HDFs to start fast cellular reprogramming including activation of stress-related kinase signaling pathways, subsequent chromatin remodeling, and expression of pluripotent-related genes via transient membrane damage caused by ultrasound-induced cavitation. And then, pluripotent markers were transported into their adjacent HDFs via direct cell-to-cell connections in order to generate xpotent clusters. The features of x/Entr cells were intermediate between pluripotency and multipotency in terms of pluripotency with three germ layer markers, multi-lineage differentiation potential, and no teratoma formation. This physical stimulus-mediated reprogramming strategy was cost-effective, simple, quick, produced significant yields, and was safe, and can therefore provide a new paradigm for clinical application.

Bioimaging of multiple piRNAs in a single breast cancer cell using molecular beacons.

Simultaneous bioimaging of piR-36026 and piR-36743 using molecular beacons successfully visualized 4 different subtypes of breast cancer.

Multiplex bioimaging of piRNA molecular pathway-regulated theragnostic effects in a single breast cancer cell using a piRNA molecular beacon.

Recently, PIWI-interacting small non-coding RNAs (piRNAs) have emerged as novel cancer biomarkers candidate because of their high expression level in various cancer types and role in the control of tumor suppressor genes. In this study, a novel breast cancer theragnostics probe based on a single system targeting the piRNA-36026 (piR-36026) molecular pathway was developed using a piR-36026 molecular beacon (MB). The piR-36026 MB successfully visualized endogenous piR-36026 biogenesis, which is highly expressed in MCF7 cells (a human breast cancer cell line), and simultaneously inhibited piR-36026-mediated cancer progression in vitro and in vivo. We discovered two tumor suppressor proteins, SERPINA1 and LRAT, that were directly regulated as endogenous piR-36026 target genes in MCF7 cells. Furthermore, multiplex bioimaging of a single MCF7 cell following treatment with piR-36026 MB clearly visualized the direct molecular interaction of piRNA-36026 with SERPINA1 or LRAT and subsequent molecular therapeutic responses including caspase-3 and PI in the nucleus.

Attenuation of Postischemic Genomic Alteration by Mesenchymal Stem Cells: a Microarray Study.

Intravenous administration of mesenchymal stem cells (IV-MSC) protects the ischemic rat brain in a stroke model, but the molecular mechanism underlying its therapeutic effect is unclear. We compared genomic profiles using the mRNA microarray technique in a rodent stroke model. Rats were treated with 1 × 10(6) IV-MSC or saline (sham group) 2 h after transient middle cerebral artery occlusion (MCAo). mRNA microarray was conducted 72 h after MCAo using brain tissue from normal rats (normal group) and the sham and MSC groups. Predicted pathway analysis was performed in differentially expressed genes (DEGs), and functional tests and immunohistochemistry for inflammation-related proteins were performed. We identified 857 DEGs between the sham and normal groups, with the majority of them (88.7%) upregulated in sham group. Predicted pathway analysis revealed that cerebral ischemia activated 10 signaling pathways mainly related to inflammation and cell cycle. IV-MSC attenuated the numbers of dysregulated genes in cerebral ischemia (118 DEGs between the MSC and normal groups). In addition, a total of 218 transcripts were differentially expressed between the MSC and sham groups, and most of them (175/218 DEGs, 80.2%) were downregulated in the MSC group. IV-MSC reduced the number of Iba-1(+) cells in the peri-infarct area, reduced the overall infarct size, and improved functional deficits in MCAo rats. In conclusion, transcriptome analysis revealed that IV-MSC attenuated postischemic genomic alterations in the ischemic brain. Amelioration of dysregulated inflammation- and cell cycle-related gene expression in the host brain is one of the molecular mechanisms of IV-MSC therapy for cerebral ischemia.

Red Blood Cell Distribution Width Is Associated with Severity of Leukoaraiosis.

Red blood cell distribution width (RDW) is one of the routine hematologic parameters reported in the complete blood count test, which has been recognized as strong prognostic marker for various medical conditions, especially cardiovascular disease. We evaluated that RDW was also associated with the leukoaraiosis; common radiological finding of brain and that has been strongly associated with risk of stroke and dementia. In the present study, we included 1006 non-stroke individuals who underwent brain MRI and routine complete blood count test including RDW. Fazekas scale was used to measure the severity of leukoaraiosis based on fluid-attenuated inversion recovery image, and the severity was dichotomized to mild-degree (Fazekas scale: 0-1) and severe-degree leukoaraiosis (Fazekas scale: 2-3). Univariate and multivariate logistic regression models were constructed to evaluate independent risk factor for severe-degree of leukoaraiosis. Mean age of 1006 subjects was 64.34 ± 9.11 year, and mean of RDW was 12.97 ± 0.86%. The severe-degree of leukoaraiosis (Fazekas scale ≥ 2) was found in 28.83%. In the multivariate logistic regression, 4th quartile of RDW (> 13.3%) were significantly associated with the presence of severe-degree of leukoaraiosis (adjusted odds ratio, 1.87; 95% confidence interval, 1.20-2.92) compared to the 1st quartile of RDW (< 12.5%). The significance was not changed after adjustments for hemoglobin and other hematologic indices. These findings suggest that RDW is independently associated with severity of leukoaraiosis.

Theragnosis-based combined cancer therapy using doxorubicin-conjugated microRNA-221 molecular beacon.

Recently, microRNA (miRNA or miR) has emerged as a new cancer biomarker because of its high expression level in various cancer types and its role in the control of tumor suppressor genes. In cancer studies, molecular imaging and treatment based on target cancer markers have been combined to facilitate simultaneous cancer diagnosis and therapy. In this study, for combined therapy with diagnosis of cancer, we developed a doxorubicin-conjugated miR-221 molecular beacon (miR-221 DOXO MB) in a single platform composed of three different nucleotides: miR-221 binding sequence, black hole quencher 1 (BHQ1), and doxorubicin binding site. Imaging of endogenous miR-221 was achieved by specific hybridization between miR-221 and the miR-221 binding site in miR-221 DOXO MB. The presence of miR-221 triggered detachment of the quencher oligo and subsequent activation of a fluorescent signal of miR-221 DOXO MB. Simultaneous cancer therapy in C6 astrocytoma cells and nude mice was achieved by inhibition of miRNA-221 function that downregulates tumor suppressor genes. The detection of miR-221 expression and inhibition of miR-221 function by miR-221 DOXO MB provide the feasibility as a cancer theragnostic probe. Furthermore, a cytotoxic effect was induced by unloading of doxorubicin intercalated into miR-221 DOXO MB inside cells. Loss of miR-221 function and cytotoxicity induced by the miR-221 DOXO MB provides combined therapeutic efficacy against cancers. This method could be used as a new theragnostic probe with enhanced therapy to detect and inhibit many cancer-related miRNAs.

Multimodal imaging probe for targeting cancer cells using uMUC-1 aptamer.

For adequate cancer therapy, newer imaging modalities with more specific ligands for unique targets are crucial. Underglycosylated mucin-1 (uMUC-1) antigen is an early marker of tumor development and is widely overexpressed on most tumors. A combination of nanotechnology with optical, radionuclide, and magnetic resonance (MR) imaging has great potential to improve cancer diagnosis and therapy. In this study, a multimodal nanoparticle imaging system was developed that can be used for optical, MR and positron emission tomography (PET) imaging. Cobalt ferrite magnetic nanoparticles surrounded by fluorescent rhodamine (designated MF) within a silica shell matrix were conjugated with an aptamer targeting uMUC-1 (designated MF-uMUC-1) and further labeled by (68)Ga (designated MFR-uMUC-1) with the help of a p-SCN-bn-NOTA chelating agent, resulting in single multimodal nanoparticles. The resultant nanoparticles are spherical and monodispersed, as revealed by transmission electron microscopy. The MFR-uMUC-1 nanoparticle showed specific and dose-dependent fluorescent, radioisotope and MR signals targeting BT-20 cells expressing uMUC-1. In vivo targeting and multimodal imaging in tumor-bearing nude mice also showed great specificity for targeting cancers with MFR-uMUC-1. The MFR-uMUC-1 probe could be used as a single multimodal probe to visualize cancer cells by means of optical, radionuclide and MR imaging.

let-7b suppresses apoptosis and autophagy of human mesenchymal stem cells transplanted into ischemia/reperfusion injured heart 7by targeting caspase-3.

INTRODUCTION: Mesenchymal stem cells (MSCs) have therapeutic potential for the repair of myocardial injury. The efficacy of MSC therapy for myocardial regeneration mainly depends on the survival of cells after transplantation into the infarcted heart. In the transplanted regions, reactive oxygen species (ROS) can cause cell death, and this process depends on caspase activation and autophagosome formation. METHODS: A Software TargetScan was utilized to search for microRNAs (miRNAs) that target caspase-3 mRNA. Six candidate miRNAs including let-7b were selected and transfected into human MSCs in vitro. Expression of MEK-EKR signal pathways and autophagy-related genes were detected. Using ischemia/reperfusion model (I/R), the effect of MSCs enriched with let-7b was determined after transplantation into infarcted heart area. Miller catheter was used to evaluate cardiac function. RESULTS: Here, we report that let-7b targets caspase-3 to regulate apoptosis and autophagy in MSCs exposed to ROS. Let-7b-transfected MSCs (let-7b-MSCs) showed high expression of survival-related proteins, including p-MEK, p-ERK and Bcl-2, leading to a decrease in Annexin V/PI- and TUNEL-positive cells under ROS-rich conditions. Moreover, autophagy-related genes, including Atg5, Atg7, Atg12 and beclin-1, were significantly downregulated in let-7b-MSCs. Using a rat model of acute myocardial infarction, we found that intramyocardial injection of let-7b-MSCs markedly enhanced left ventricular (LV) function and microvessel density, in accordance with a reduced infarct size and the expression of caspase-3. CONCLUSIONS: Taken together, these data indicate that let-7b may protect MSCs implanted into infarcted myocardium from apoptosis and autophagy by directly targeting caspase-3 signaling.

Sperm DNA-mediated reduction of nonspecific fluorescence during cellular imaging with quantum dots.

Salmon sperm DNA was used as a blocking agent to reduce background fluorescence signals from gelatin-coated cell culture dishes.

Quantum dot-based molecular beacon to monitor intracellular microRNAs.

Fluorescence monitoring of endogenous microRNA (miRNA or miR) activity related to neuronal development using nano-sized materials provides crucial information on miRNA expression patterns in a noninvasive manner. In this study, we report a new method to monitor intracellular miRNA124a using quantum dot-based molecular beacon (R9-QD-miR124a beacon). The R9-QD-miR124a beacon was constructed using QDs and two probes, miR124a-targeting oligomer and arginine rich cell-penetrating peptide (R9 peptide). The miR124a-targeting oligomer contains a miR124a binging sequence and a black hole quencher 1 (BHQ1). In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon. The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal. Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

Simultaneous imaging of two different cancer biomarkers using aptamer-conjugated quantum dots.

Studying gene expression profile in a single cancer cell is important because multiple genes are associated with cancer development. Quantum dots (QDs) have been utilized as biological probes for imaging and detection. QDs display specific optical and electrical properties that depend on their size that can be applied for imaging and sensing applications. In this study, simultaneous imaging of the cancer biomarkers, tenascin-C and nucleolin, was performed using two types of aptamer-conjugated QDs. The simultaneous imaging of these two different cancer markers in three cancer cell lines was reliable and cell line-specific. Current requirements for cancer imaging technologies include the need for simple preparation methods and the ability to detect multiple cancer biomarkers and evaluate their intracellular localizations. The method employed in this study is a feasible solution to these requirements.

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.

Real-time imaging of the epithelial-mesenchymal transition using microRNA-200a sequence-based molecular beacon-conjugated magnetic nanoparticles.

The epithelial-mesenchymal transition (EMT) plays important roles in tumor progression to metastasis. Thus, the development of an imaging probe that can monitor transient periods of the EMT process in live cells is required for a better understanding of metastatic process. Inspired by the fact that the mRNA expression levels of zinc finger E-box-binding homeobox 1 (ZEB1) increase when cells adopt mesenchyme characteristics and that microRNA-200a (miR-200a) can bind to ZEB1 mRNA, we conjugated molecular beacon (MB) mimicking mature miR-200a to magnetic nanoparticles (miR-200a-MB-MNPs) and devised an imaging method to observe transitional changes in the cells during EMT. Transforming growth factor-ß1 treated epithelial cells and breast cancer cell lines representing both epithelial and mesenchymal phenotypes were used for the validation of miR-200a-MB-MNPs as an EMT imaging probe. The real-time imaging of live cells acquired with the induction of EMT revealed an increase in fluorescence signals by miR-200a-MB-MNPs, cell morphology alterations, and the loss of cell-cell adhesion. Our results suggest that miR-200a-MB-MNPs can be used as an imaging probe for the real-time monitoring of the EMT process in live cells.

A color-tunable molecular beacon to sense miRNA-9 expression during neurogenesis.

A typical molecular beacon (MB) composing of a fluorophore and a quencher has been used to sense various intracellular biomolecules including microRNAs (miRNA, miR). However, the on/off-tunable miRNA MB is difficult to distinguish whether the observed low fluorescence brightness results from low miRNA expression or low transfection of the miRNA MB. We developed a color-tunable miRNA-9 MB (ColoR9 MB) to sense miR-9 expression-dependent color change. The ColoR9 MB was synthesized by a partially double-stranded DNA oligonucleotide containing a miR-9 binding site and a reporter probe with Cy3/black hole quencher 1 (BHQ1) at one end and a reference probe with Cy5.5 at the other end. The ColoR9 MB visualized CHO and P19 cells with red color in the absence of miR-9 and yellow color in the presence of miR-9. In vivo imaging demonstrated that the green fluorescence recovery of the reporter probe from the ColoR9 MB increased gradually during neuronal differentiation of P19 cells, whereas red fluorescence activity of the reference probe remained constant. These results showed the great specificity of sensing miR-9 expression- and neurogenesis-dependent color change.

Bioluminescence reporter gene-based detection of microRNAs.

MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that inhibit the expression of cognate genes in multicellular organisms. These small RNAs have been demonstrated to play crucial roles in a variety of biological processes including cell differentiation, proliferation, and survival. Knowledge of specific expression patterns of miRNAs is critical for functional studies. Here, we describe a bioluminescence reporter gene-based method to measure miRNA activity in cultured cells and mice using a Gaussia luciferase reporter gene controlled by miRNA binding sites in its 3'untranslated region. This method can be used to noninvasively monitor the expression patterns of functionally active miRNAs involved in different biological processes or diseases in mice.

In vivo imaging of tumour bearing near-infrared fluorescence-emitting carbon nanodots derived from tire soot.

NIRF imaging of carbon nanodots derived from tire soot was clearly visualized in glioma in vitro and in vivo.

Bioimaging of targeting cancers using aptamer-conjugated carbon nanodots.

Using the excellent emission properties of carbon nanodots, we have developed an aptamer-conjugated imaging probe for targeting cancers.

Consecutive targetable smart nanoprobe for molecular recognition of cytoplasmic microRNA in metastatic breast cancer.

We report smart nanoprobe, hyaluronic acid (HA)-based nanocontainers containing miR-34a beacons (bHNCs), for the intracellular recognition of miR-34a levels in metastatic breast cancer cells, which is distinct from the imaging of biomarkers such of cell membrane receptors such as HER2. In this study, we demonstrate that a nanoscale vesicle that couples a targeting endocytic route, CD44, and a molecular imaging probe enables the efficient detection of specific miRNAs. Furthermore, bHNCs showed no cytotoxicity and high stability due to the anchored HA molecules on the surface of nanocontainers, and enables the targeted delivery of beacons via CD44 receptor-mediated endocytosis. In vitro and in vivo optical imaging using bHNCs also allow the measurement of miR-34a expression levels due to the selective recognition of the beacons released from the internalized bHNCs. We believe that the technique described herein can be further developed as a cancer diagnostic as well as a miRNA-based therapy of metastatic cancer.

VEGF receptor binding peptide-linked amphiphilic peptide with arginines and valines for endothelial cell-specific gene delivery.

An amphiphilic peptide with a 3-arginine stretch and a 6-valine stretch (R3V6) has been previously reported to deliver plasmid DNA (pDNA) into cells with no toxicity. Here, the vascular endothelial growth factor receptor binding peptide (VRBP) was linked to R3V6 to promote endothelial-specific gene delivery. The pDNA/VRBP-linked R3V6 (VRBP-R3V6) complex was physically characterized via various methods. In a gel retardation assay, pDNA was completely retarded by VRBP-R3V6 at a weight ratio of 1:2 (pDNA:peptide). VRBP-R3V6 also protected pDNA from DNase I for longer than 60 min. Heparin competition assay showed that the pDNA/VRBP-R3V6 complex did not release pDNA when heparin was introduced at a two-fold weight excess of pDNA. In vitro transfection showed that VRBP-R3V6 had transfection efficiency into endothelial cells approximately 200 times greater than that of R3V6. In addition, the transfection efficiency was further enhanced into hypoxic endothelial cells. However, in human embryonic kidney 293 and neuroblastoma N2A cells, VRBP-R3V6 only achieved a transfection rate 10 times higher than R3V6, indicating that VRBP-R3V6 has high specificity for endothelial cells. VRBP-R3V6 was also shown to be nontoxic in a cytotoxicity assay. The data presented here suggest that VRBP-R3V6 may prove useful for specific gene delivery to endothelial cells.