1,717 research outputs found
Cytoplasmic RNA in undifferentiated neural stem cells: a potential label-free Raman spectral marker for assessing the undifferentiated status
Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ~700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (~1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA
Cytoplasmic RNA in undifferentiated neural stem cells: A potential label-free raman spectral marker for assessing the undifferentiated status
Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ∼700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (∼1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA. © 2012 American Chemical Society
Hydrogel-Based Colorimetric Assay for Multiplexed MicroRNA Detection in a Microfluidic Device
Although microRNA (miRNA) expression levels provide important information regarding disease states owing to their unique dysregulation patterns in tissues, translation of miRNA diagnostics into point-of-care (POC) settings has been limited by practical challenges. Her; we developed a hydrogel-based microfluidic platform for colorimetric profiling of miRNAs, without the use of complex external equipment for fluidics and imaging. For sensitive and reliable measurement without the risk of sequence bias, we employed a gold deposition-based signal amplification scheme and dark-field imaging, and seamlessly integrated a previously developed miRNA assay scheme into this platform. The assay demonstrated a limit of detection of 260 fM, along with multiplexing of small panels of miRNAs in healthy and cancer samples. We anticipate this versatile platform to facilitate a broad range of POC profiling of miRNAs in cancer-associated dysregulation with high-confidence by exploiting the unique features of hydrogel substrate in an on-chip format and colorimetric analysis
Highly specific plasmonic biosensors for ultrasensitive microRNA detection in plasma from pancreatic cancer patients
MicroRNAs (miRs) are small noncoding RNAs that regulate mRNA stability and/or translation. Because of their release into the circulation and their remarkable stability, miR levels in plasma and other biological fluids can serve as diagnostic and prognostic disease biomarkers. However, quantifying miRs in the circulation is challenging due to issues with sensitivity and specificity. This Letter describes for the first time the design and characterization of a regenerative, solid-state localized surface plasmon resonance (LSPR) sensor based on highly sensitive nanostructures (gold nanoprisms) that obviates the need for labels or amplification of the miRs. Our direct hybridization approach has enabled the detection of subfemtomolar concentration of miR-X (X = 21 and 10b) in human plasma in pancreatic cancer patients. Our LSPR-based measurements showed that the miR levels measured directly in patient plasma were at least 2-fold higher than following RNA extraction and quantification by reverse transcriptase-polymerase chain reaction. Through LSPR-based measurements we have shown nearly 4-fold higher concentrations of miR-10b than miR-21 in plasma of pancreatic cancer patients. We propose that our highly sensitive and selective detection approach for assaying miRs in plasma can be applied to many cancer types and disease states and should allow a rational approach for testing the utility of miRs as markers for early disease diagnosis and prognosis, which could allow for the design of effective individualized therapeutic approaches
The Role of lncRNAs TAPIR-1 and -2 as Diagnostic Markers and Potential Therapeutic Targets in Prostate Cancer
In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA (TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa
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Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses.
Extracellular RNAs (exRNAs) can be released by numerous cell types in vitro, are often protected within vesicles, and can modify recipient cell function. To determine how the composition and cellular sources of exRNAs and the extracellular vesicles (EVs) that carry them change in vivo during tissue inflammation, we analyzed bronchoalveolar lavage fluid (BALF) from mice before and after lung allergen challenge. In the lung, extracellular microRNAs (ex-miRNAs) had a composition that was highly correlated with airway-lining epithelium. Using cell type-specific membrane tagging and single vesicle flow, we also found that 80% of detected vesicles were of epithelial origin. After the induction of allergic airway inflammation, miRNAs selectively expressed by immune cells, including miR-223 and miR-142a, increased and hematopoietic-cell-derived EVs also increased >2-fold. These data demonstrate that infiltrating immune cells release ex-miRNAs and EVs in inflamed tissues to alter the local extracellular environment
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The Mechanistic Role of Long Non-coding RNAs in Advanced Prostate Cancer Response to Therapy
Prostate cancer is one of the most common malignancies and the fifth leading cause of cancer related deaths in males, worldwide. It is commonly treated using androgen deprivation therapy (ADT), but around 25% develop ADT resistance and are called Castration-Resistant Prostate Cancers (CRPCs). Therapies currently used for CRPC treatment include: the new generation anti-androgen enzalutamide, the taxane cabazitaxel and carboplatin. Despite the prolonged survival resulting from these treatments, CRPC is still incurable. Recent evidence suggests that long non-coding RNAs (lncRNAs) promote drug resistance. The lncRNA HORAS5 (i.e. linc00161) regulates drug response in different cancers and is upregulated in CRPC versus hormone-sensitive patient-derived xenografts (PDXs), thereby stimulating pro-survival mechanisms. This project has investigated whether HORAS5 has a role in CRPC response to therapy. CRPC cells have been treated with different concentrations of cabazitaxel, carboplatin and enzalutamide. Cabazitaxel exposure increases HORAS5 expression, in androgen receptor-positive (AR+) and -negative (AR–) prostate cancer cells and HORAS5 overexpression decreases cabazitaxel sensitivity and cell apoptosis. HORAS5 RNA interference (RNAi) increases cabazitaxel sensitivity and cell apoptosis. Next-generation RNA sequencing and real time qPCR have shown that the anti-apoptotic factor BCL2A1 is significantly upregulated upon HORAS5 overexpression in AR- prostate cancer cells exposed to cabazitaxel. BCL2A1 silencing decreases cell count and increases apoptosis of prostate cancer cells exposed to cabazitaxel. HORAS5 and BCL2A1 upregulation is associated with decreased survival in prostate cancer patients and HORAS5 is upregulated in clinical samples from prostate cancer patients exposed to taxanes. Transfection of CRPC cells with HORAS5-targeting antisense oligonucleotides (ASOs) efficiently reduces HORAS5 expression, thereby decreasing cabazitaxel IC50 when tested in combination with this drug. Overall, this project shows that HORAS5 stimulates BCL2A1 expression in prostate cancer cells, thereby reducing caspase-mediated apoptosis. This leads to increased cabazitaxel resistance. The clinical relevance of HORAS5 expression and the effect of HORAS5-targeting ASOs shown in this thesis highlight the translational potential of HORAS5 modulation. This work sheds light on the relevance of lncRNAs in cancer drug resistance and proposes the use of HORAS5 as a future therapeutic target to increase therapy efficacy in CRPC
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