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.

Generation of directly reprogrammed human endothelial cells derived from fibroblast using ultrasound.

Physical microenvironment plays an important role in determining cellular reprogramming. In this study, we first generated directly reprogrammed human dermal fibroblasts (HDFs) into endothelial cells (ECs) mediated by environmental transition-guided cellular reprogramming (e/Entr) using ultrasound and characterized e/Entr. Ultrasound stimulus was introduced to ECs culture media and HDFs and induced into ECs-like cells. We performed microarray, RT-PCR, protein analysis, matrigel plug assay and e/Entr were transplanted into ischemic hindlimb mice model. Here we show that the activation of MAPK signaling pathways and the modulation of histone proteins such as Hp1-α, H3K27me3 and H3K4me3 in e/Entr contribute to the changes in chromatin configuration and reprogramming. Microarray data demonstrated that e/Entr highly expressed genes associated with ECs transcription factors and angiogenesis. In addition, the transplantation of e/Entr into hindlimb ischemia showed a high recovery of blood perfusion, limb salvage and e/Entr contributed to the formation of new vessels. In conclusion, the present study provided the first evidence that ultrasound reprogramming can induce postnatal cells to functional ECs. Therefore, our data suggest that physical stimulus-mediated reprogramming is a highly effective and safe strategy for the novel therapeutic alternatives.

Exosome-Mediated Ultra-Effective Direct Conversion of Human Fibroblasts into Neural Progenitor-like Cells.

Exosomes, naturally secreted nanoparticles, have been introduced as vehicles for horizontal transfer of genetic material. We induced autologous exosomes containing a cocktail of reprogramming factors ("reprosomes") to convert fibroblasts into neural progenitor cells (NPCs). The fibroblasts were treated with ultrasound and subsequently cultured in neural stem cell medium for 1 day to induce the release of reprosomes composed of reprogramming factors associated with chromatin remodeling and neural lineage-specific factors. After being treated with reprosomes, fibroblasts were converted into NPCs (rNPCs) with great efficiency via activation of chromatin remodeling, so quickly that only 5 days were required for the formation of 1500 spheroids showing an NPC-like phenotype. The rNPCs maintained self-renewal and proliferative properties for several weeks and successfully differentiated into neurons, astrocytes, and oligodendrocytes in vitro and in vivo. Reprosome-mediated cellular reprogramming is simple, safe, and efficient to produce autologous stem cells for clinical application.

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.

Molecular Beacon-Based MicroRNA Imaging During Neurogenesis.

The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quenched by BHQ2 was gradually recovered as increasing concentration of the miR-124a in tube. The functional work of miR-124a beacon was examined in intracellular environment, allowing for the internalization of the miR-124a beacon by lipofectamine, which resulted in activated fluorescent signals of the miR-124a beacon in the HeLa cells after the addition of synthetic miR-124a. The endogenous miR-124a expression level was detected by miR-124a beacon system during neurogenesis, showing brighter fluorescence intensity in cytoplasmic area of P19 cells after induction of neuronal differentiation by retinoic acid. The molecular beacon based-miRNA detection technique could be applicable to the simultaneous visualization of a variety of miRNA expression patterns using different fluorescence dyes. For the study of examining endogenous miRNA expression level using miRNA-beacon system, if cellular differentiation step is already prepared, transfection step of miR-124a beacon into P19 cells, and acquisition of activated fluorescence signal measured by confocal microscope can be conducted approximately within 6 h.

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.

Association between Serum Alkaline Phosphatase Level and Cerebral Small Vessel Disease.

BACKGROUND: Serum alkaline phosphatase (ALP) is a marker of vascular calcification. A high serum ALP level is associated with an increase in cardiovascular events, and predicts poor functional outcome in patients with stroke. We investigated whether serum ALP was associated with cerebral small vessel disease (cSVD) and large cerebral artery stenosis (LCAS). METHODS: We evaluated vascular risk factors, brain magnetic resonance images (MRIs), and MR angiograms from 1,011 neurologically healthy participants. The presence of silent lacunar infarction (SLI) and moderate-to-severe cerebral white matter hyperintensities (MS-cWMH) were evaluated as indices of cSVD on brain MRIs. Findings of extracranial arterial stenosis (ECAS) or intracranial arterial stenosis (ICAS) were considered to be indices of LCAS on MR angiograms. RESULTS: Subjects with SLI (odds ratio [OR]: 2.09; 95% confidence interval [CI]: 1.27-3.42; p = 0.004) and MS-cWMH (OR: 1.48; 95% CI; 1.03-2.13, p = 0.036) were significantly more likely to have ALP levels in the third tertile (ALP ≥ 195 IU/L) than the first tertile (ALP ≤ 155 IU/L), after adjusting for cardiovascular risk factors. The mean serum ALP level was significantly higher in patients with SLI or MS-cWMH compared to patients without those findings. After adjustment for confounding factors, the multivariate model found that the statistical significance of serum ALP remained when the presence of SLI (OR: 1.05 per 10 IU/L increase in ALP; 95% CI: 1.02-1.08; p = 0.003) or MS-cWMH (OR: 1.03 per 10 IU/L increase in ALP; 95% CI: 1.00-1.06; p = 0.025) were added to the model. There were no differences in the proportions of patients with LCAS, ICAS, and ECAS across the serum ALP tertiles. CONCLUSIONS: Our study of neurologically healthy participants found a positive association between serum ALP level and indicators of cSVD, but no association between serum ALP level and the indicators of LCAS.

Bioimaging of microRNA34c in a single sperm using a molecular beacon.

The VisuFect-conjugated molecular beacon was developed for non-invasive visualization of microRNA34c in a living single mouse sperm.

VisuFect-mediated siRNA delivery into zygotes.

Current methods for delivering foreign genetic materials into mammalian cells are highly successful. However, these methods cannot be applied in oocyte or embryo systems due to their toxicity and low efficiency. Moreover, no satisfactory methods exist for delivering foreign genetic material without inducing physical damage to membranes. Here we developed an organic compound (VisuFect)-mediated small interfering RNA (siRNA) delivery method and evaluated this method in P19 cells and mouse zygotes. Oct4-siRNA conjugated VisuFect (Oct4-siRNA-VF) permeated the zona pellucida effectively and localized inside mouse zygotes without inducing membrane damage. Successful VisuFect-mediated delivery was further demonstrated by strong transcriptional repression of Oct4 expression by the delivered Oct4-siRNA, in addition to repressed embryonic development of mouse zygotes.

Bioimaging of microRNA124a-independent neuronal differentiation of human G2 neural stem cells.

Evaluation of the function of microRNAs (miRNAs or miRs) through miRNA expression profiles during neuronal differentiation plays a critical role not only in identifying unique miRNAs relevant to cellular development but also in understanding regulatory functions of the cell-specific miRNAs in living organisms. Here, we examined the microarray-based miRNA expression profiles of G2 cells (recently developed human neural stem cells) and monitored the expression pattern of known neuron-specific miR-9 and miR-124a during neuronal differentiation of G2 cells in vitro and in vivo. Of 500 miRNAs analyzed by microarray of G2 cells, the expression of 90 miRNAs was significantly increased during doxycycline-dependent neuronal differentiation of G2 cells and about 60 miRNAs showed a gradual enhancement of gene expression as neuronal differentiation progressed. Real-time PCR showed that expression of endogenous mature miR-9 was continuously and gradually increased in a pattern dependent on the period of neuronal differentiation of G2 cells while the increased expression of neuron-specific mature miR-124a was barely observed during neurogenesis. Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR-9 and CMV/Gluc/3×PT_miR-124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR-9 was gradually decreased both in vitro and in vivo in G2 cells induced to differentiate into neurons. However, in vitro and in vivo bioluminescence signals for CMV/Gluc/3×PT_miR-124a were not significantly different between undifferentiated and differentiated G2 cells. Our results demonstrate that biogenesis of neuron-specific miR-124a is not necessary for doxycycline-dependent neurogenesis of G2 cells.

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.

Bioimaging of transcriptional activity of microRNA124a during neurogenesis.

OBJECTIVES: A special vector system was developed to monitor the in vitro and in vivo endogenous level of a primary transcript of miR124a during neuronal differentiation RESULTS: The upstream regions of miR124a were fused with luciferase (Gluc) and their activity was measured. During neurogenesis of P19 cells, the primary transcript level of miR124a was increased 1.5-times compared to the undifferentiated P19 cells. P19 cells grafted to nude mice exhibited the same pattern of luciferase activity in vivo as they did in vitro. CONCLUSION: The expression of primary miR124a during neurogenesis was successfully imaged by in vitro and in vivo luciferase reporter gene-based method.

SPECT/CT Imaging of High-Risk Atherosclerotic Plaques using Integrin-Binding RGD Dimer Peptides.

Vulnerable atherosclerotic plaques with unique biological signatures are responsible for most major cardiovascular events including acute myocardial infarction and stroke. However, current clinical diagnostic approaches for atherosclerosis focus on anatomical measurements such as the degree of luminal stenosis and wall thickness. An abundance of neovessels with elevated expression of integrin αvß3 is closely associated with an increased risk of plaque rupture. Herein we evaluated the potential of an αvß3 integrin-targeting radiotracer, (99m)Tc-IDA-D-[c(RGDfK)]2, for SPECT/CT imaging of high-risk plaque in murine atherosclerosis models. In vivo uptake of (99m)Tc-IDA-D-[c(RGDfK)]2 was significantly higher in atherosclerotic aortas than in relatively normal aortas. Comparison with the negative-control peptide, (99m)Tc-IDA-D-[c(RADfK)]2, proved specific binding of (99m)Tc-IDA-D-[c(RGDfK)]2 for plaque lesions in in vivo SPECT/CT and ex vivo autoradiographic imaging. Histopathological characterization revealed that a prominent SPECT signal of (99m)Tc-IDA-D-[c(RGDfK)]2 corresponded to the presence of high-risk plaques with a large necrotic core, a thin fibrous cap, and vibrant neoangiogenic events. Notably, the RGD dimer based (99m)Tc-IDA-D-[c(RGDfK)]2 showed better imaging performance in comparison with the common monomeric RGD peptide probe (123)I-c(RGDyV) and fluorescence tissue assay corroborated this. Our preclinical data demonstrated that (99m)Tc-IDA-D-[c(RGDfK)]2 SPECT/CT is a sensitive tool to noninvasively gauge atherosclerosis beyond vascular anatomy by assessing culprit plaque neovascularization.

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.