Absence Of microRNAs Research Articles

Catalpol inhibits the proliferation, migration and metastasis of HCC cells by regulating miR‑140‑5p expression.

Hepatocellular carcinoma (HCC) is a frequent malignant tumor. Catalpol is a Chinese medicine extract with a number of pharmacologically active properties. The present study aimed to investigate the effects and mechanisms of catalpol in HCC. HCC cells were treated with catalpol in the presence or absence of microRNA (miR)-140-5p inhibitor, and assays to determine cell viability, proliferation, invasion and migration were performed. Reverse transcription-quantitative PCR and western blotting were performed to determine the mRNA and protein expression levels of miR-140-5p, vimentin, N-Cadherin and E-Cadherin. Moreover, cells were treated with catalpol in the absence or presence of transforming growth factor (TGF)-β1, and the cell morphology was observed under a microscope. The results demonstrated that catalpol inhibited cell proliferation, invasion and migration, and decreased the expression levels of vimentin and N-cadherin, but increased the expression levels of E-cadherin and miR-140-5p. Catalpol inhibited morphological changes in epithelial-mesenchymal transformation (EMT) of cells induced by TGF-β1. Following inhibition of miR-140-5p expression, the proliferation, invasion and migration of HCC cells were promoted, E-cadherin expression was decreased, and the levels of vimentin and N-cadherin were increased. The miR-140-5p inhibitor effectively reversed the inhibitory effect of catalpol on cell proliferation, invasion and migration. Thus, the results suggested that the antitumor potential of catalpol in HCC may be exerted by regulating the expression of miR-140-5p to inhibit proliferation, invasion, migration and EMT of HCC cells.

A validated miRNA signature for the diagnosis of osteoporosis related fractures using SVM algorithm classification.

The aim of the present study was to develop a circulating microRNA expression signature for early prediction of osteoporotic fractures and to validate the results using Gene Expression Omnibus (GEO) datasets. The GSE70318 dataset was downloaded from GEO and used to build an osteoporotic fracture prediction model based on the receiver operating characteristic curve and support vector machine (SVM) classification index. The GSE74209 dataset was used as a validation dataset. Additionally, in vitro, alkaline phosphatase (ALP) activity was measured in the presence or absence of microRNA (miRNA/miR) treatments in human osteoblast cells. The expression of two selected genes was detected by western blotting. miR-188-3p, miR-942-3p, miR-576-3p and miR-135a-5p were differentially expressed between controls and osteoporotic patients with fractures. SVM classification using these four miRNAs provided better dichotomization. It was further confirmed that miR-576-3p and 135a-5p in the GSE74209 dataset could also significantly discriminate between the controls and fracture patients, the area under the curve of SVM2 was 0.9722 with 95% CI 0.8885-1.056. Further analysis indicated that the target genes of the two miRNAs participated in the Wingless-related integration site, Hedgehog and transforming growth factor-β signaling pathways and osteoclast differentiation. miR-576-3p and miR-135-5p transfection decreased ALP activity and ALP activity was increased in the presence of blocking antisense oligonucleotides. Western blotting indicated miR-576-3p and miR-135-5p decreased CSNK1A1L and LRP6 levels, respectively. In conclusion, two miRNA signatures were developed and validated for the prediction of osteoporotic fractures.

MoS2 quantum dots modified with a labeled molecular beacon as a ratiometric fluorescent gene probe for FRET based detection and imaging of microRNA.

A dual-channel ratiometric nanoprobe is described for detection and imaging of microRNA. It was prepared from MoS2 quantum dots (QDs; with blue emission and excitation/emission peaks at 310/398nm) which acts as both the gene carrier and as a donor in fluorescence resonance energy transfer (FRET). Molecular beacons containing loops for molecular recognition of microRNA and labeled with carboxyfluorescein (FAM)were covalently attached to the MoS2 QDs and serve as the FRET acceptor. In the absence of microRNA, the nanoprobe exhibits low FRET efficiency due to the close distance between the FAM tag and the QDs. Hybridization with microRNA enlarges the distance between QD and beacon. This results in an enhancement of the FRET efficiency of the nanoprobe. The ratio of green and blue fluorescence (I520/I398) increases linearly in the 5 to 150nM microRNA concentration range in both aqueous solution and diluted artificial cerebrospinal fluid. The limit of detection (LOD) is as low as 0.38nM and 0.52nM, respectively. Other features of this nanoprobe include (a) excellent resistance to nuclease-induced false positive signals and (b) the option to use it for distinguishing different cell lines by in-situ imaging of intracellular microRNAs. Graphical abstract Schematic of a dual-channel photoluminescence nanoprobe for the determination of microRNA-21 (miR-21) by monitoring the microRNA-triggered enhancement of the fluorescence resonance energy transfer (FRET) efficiency between MoS2 QDs and carboxyfluorescein-labeled molecular beacons.

Bim gene dosage is critical in modulating nephron progenitor survival in the absence of microRNAs during kidney development.

Low nephron endowment at birth has been associated with an increased risk for developing hypertension and chronic kidney disease. We demonstrated in an earlier study that conditional deletion of the microRNA (miRNA)-processing enzyme Dicer from nephron progenitors results in premature depletion of the progenitors and increased expression of the proapoptotic protein Bim (also known as Bcl-2L11). In this study, we generated a compound mouse model with conditional deletion of both Dicer and Bim, to determine the biologic significance of increased Bim expression in Dicer-deficient nephron progenitors. The loss of Bim partially restored the number of nephron progenitors and improved nephron formation. The number of progenitors undergoing apoptosis was significantly reduced in kidneys with loss of a single allele, or both alleles, of Bim compared to mutant kidneys. Furthermore, 2 miRNAs expressed in nephron progenitors (miR-17 and miR-106b) regulated Bim levels in vitro and in vivo Together, these data suggest that miRNA-mediated regulation of Bim controls nephron progenitor survival during nephrogenesis, as one potential means of regulating nephron endowment.-Cerqueira, D. M., Bodnar, A. J., Phua, Y. L., Freer, R., Hemker, S. L., Walensky, L. D., Hukriede, N. A., Ho, J. Bim gene dosage is critical in modulating nephron progenitor survival in the absence of microRNAs during kidney development.

Regulatory T cell microRNA expression changes in children with acute Kawasaki disease.

Kawasaki disease (KD) is a type of systemic vasculitis syndrome related to immune dysfunction. Previous studies have implicated that dysfunctional regulatory T cells (Treg ) may be associated with the immune dysfunction in KD. In the absence of microRNAs (miRNAs), forkhead box protein 3 (FoxP3)(+) Treg develop but fail to maintain immune homeostasis. This study was designed to investigate the effects of miR-155, miR-21 and miR-31 on Treg in children with KD. The proportions of CD4(+) CD25(+) FoxP3(+) Treg and the mean fluorescence intensity (MFI) of phosphorylated-signal transducer and activator of transcription (pSTAT)-5 and pSTAT-3 protein in CD4(+) CD25(+) Treg were analysed by flow cytometry. The concentration of interleukin (IL)-6 in plasma was measured by cytometric bead array. Real-time polymerase chain reaction was performed to detect the levels of microRNAs and associated factors in CD4(+) CD25(+) Treg . The proportion of Treg and the mRNA levels of the associated factors [FoxP3, glucocorticoid-induced tumour necrosis factor-receptor (GITR), cytotoxic T lymphocyte antigen (CTLA)-4)] were significantly lower in KD patients (P < 0·05). MiR-155 and miR-21 levels were significantly down-regulated and miR-31 expression was higher in KD patients (P < 0·05). Plasma interleukin (IL)-6 concentrations, pSTAT-3 protein levels and suppressors of cytokine signalling (SOCS)-1 mRNA expression were remarkably elevated in acute KD (P < 0·05), while pSTAT-5 protein levels were remarkably decreased in acute KD (P < 0·05). These findings were reversed after intravenous immunoglobulin treatment (P < 0·05). Our results demonstrate that FoxP3 mRNA levels were primarily affected by the miR-155/SOCS1 and the miR-31 signalling pathways. These results suggest that the decrease in FoxP3(+) Treg might be associated with decreased expression of miR-155, leading to aberrant SOCS1/STAT-5 signalling and overexpression of miR-31 in patients with acute KD.

Reconsidering the phylogenetic utility of miRNA in animals.

Phylogenetics, the study of evolutionary relationships, is critical to understanding the history of life on Earth, integrating and synthesizing information collected across diverse organisms, and testing hypotheses about evolutionary patterns and processes. Most phylogenetic studies are now based on detailed analyses of gene sequence evolution. Over the last several decades, these studies have provided a much better understanding of the structure of the Tree of Life. This progress has also highlighted and helped define many tough phylogenetic questions that have not been answered yet with available data and analysis methods. This has inspired the search for new categories of data that could succeed where analyses of gene sequence evolution have not yet provided strong, consistent phylogenetic support. In PNAS, Thomson et al. (1) examine one of these new categories of data (2⇓⇓–5): the presence and absence of microRNA (miRNA) families. They find that there are several reasons why they may not be as useful for estimating phylogenies as has been proposed. miRNAs are small RNA genes that are involved in posttranscriptional gene regulation (6, 7). They originated independently in plants, animals, and several other organisms (8). miRNAs and the enzymes needed to process them are missing entirely from comb jellies (ctenophores), suggesting that they may have arisen within animals rather than before the most recent common ancestor of animals (9). Different miRNA families are found in different animals, providing a potential source of information on animal relationships. The sequence of each miRNA family is highly conserved, which makes them relatively easy to tell apart. Why was it believed that investigating the presence and absence of miRNAs could succeed where other types of phylogenetic character data have failed? It comes down to homoplasy: any evolutionary pattern of character evolution that deviates from the simplest possible evolutionary … [↵][1]1Email: casey_dunn{at}brown.edu. [1]: #xref-corresp-1-1

MicroRNA-sensitive Oncolytic Measles Viruses for Cancer-specific Vector Tropism

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased tumor-cell specificity, and are currently under investigation in clinical trials including a phase I study for glioblastoma multiforme (GBM). Recent preclinical studies have shown that the cellular tropism of several viruses can be controlled by inserting microRNA-target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. Since neuron-specific microRNA-7 is downregulated in gliomas but highly expressed in normal brain tissue, we engineered a microRNA-sensitive virus containing target sites for microRNA-7 in the 3'-untranslated region of the viral fusion gene. In presence of microRNA-7 this modification inhibits translation of envelope proteins, restricts viral spread, and progeny production. Even though highly attenuated in presence of microRNA-7, this virus retained full efficacy against glioblastoma xenografts. Furthermore, microRNA-mediated inhibition protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Importantly, endogenous microRNA-7 expression in primary human brain resections tightly restricted replication and spread of microRNA-sensitive virus. This is proof-of-concept that tropism restriction by tissue-specific microRNAs can be adapted to oncolytic MV to regulate viral replication and gene expression to maximize tumor specificity without compromising oncolytic efficacy.

MicroRNA-375 and MicroRNA-221: Potential Noncoding RNAs Associated with Antiproliferative Activity of Benzyl Isothiocyanate in Pancreatic Cancer

The deregulated presence or absence of microRNAs (miRNAs) might play an important role in molecular pathways leading to neoplastic transformation. At present, it is also thought that the approaches to interfere miRNA functions should be helpful for developing novel therapeutic opportunities for human cancer. In this study, we provide evidence that the anticancer agent benzyl isothiocyanate (BITC) has the ability to modulate the level of miRNAs such as miR-221 and miR-375, known to be abnormally expressed in pancreatic cancer patients. Interestingly, ectopic expression of miR-375 or the enforced silencing of miR-221 in cultured pancreatic cancer cells attenuates cell viability and sensitizes antiproliferative action of BITC. We also show that the expression of putative tumor suppressor miR-375 is more abundant in nonpathological mice pancreata than those with Kras(G12D)-driven pancreatic intraepithelial neoplasia (PanIN). To the contrary, the expression of oncogenic miR-221 is significantly elevated in the mouse pancreas with PanIN lesions. Although miR-375 has been shown to be aberrantly expressed in pancreatic cancer patients, there has not been a comprehensive study to investigate the molecular pathways targeted by this miRNA in pancreatic cancer cells. Further analysis by gene expression microarray revealed that IGFBP5 and CAV-1, potential biomarkers of pancreatic cancer, were significantly downregulated in cells transfected with miR-375. Correlatively, elevated expression of IGFBP5 and CAV-1 was evident in the mouse pancreas with preneoplastic lesions in which the expression of miR-375 wanes. Taken together, our findings suggest that anticancer agent BITC might target the expression of miR-221 and miR-375 to switch hyperproliferative pancreatic cancer cells to a hypoproliferative state.

Reversible Block of Mouse Neural Stem Cell Differentiation in the Absence of Dicer and MicroRNAs

BackgroundTo investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs.Principal FindingsDicer-null NS cells underwent normal self-renewal and could be maintained in vitro indefinitely, but had subtly altered cell cycle kinetics and abnormal heterochromatin organisation. In the absence of all microRNAs, Dicer-null NS cells were incapable of generating either glial or neuronal progeny and exhibited a marked dependency on exogenous EGF for survival. Dicer-null NS cells assumed complex differences in mRNA and protein expression under self-renewing conditions, upregulating transcripts indicative of self-renewing NS cells and expressing genes characteristic of differentiating neurons and glia. Underlining the growth-factor dependency of Dicer-null NS cells, many regulators of apoptosis were enriched in expression in these cells. Dicer-null NS cells initiate some of the same gene expression changes as wild-type cells under astrocyte differentiating conditions, but also show aberrant expression of large sets of genes and ultimately fail to complete the differentiation programme. Acute replacement of Dicer restored their ability to differentiate to both neurons and glia.ConclusionsThe block in differentiation due to loss of Dicer and microRNAs is reversible and the significantly altered phenotype of Dicer-null NS cells does not constitute a permanent transformation. We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation.

Electrochemical Detection of MicroRNAs via Gap Hybridization Assay

MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.

Cutting Edge: The Foxp3 Target miR-155 Contributes to the Development of Regulatory T Cells

Foxp3 is a transcription factor that is essential for the normal development of regulatory T cells (Tregs). In the absence of microRNAs (miRNAs), Foxp3(+) Tregs develop but fail to maintain immune homeostasis, leading to a scurfy-like disease. Global analysis of the network of genes regulated by Foxp3 has identified the miRNA miR-155, which is highly expressed in Tregs, as a direct target of Foxp3. In this study we report that miR-155-deficient mice have reduced numbers of Tregs, both in the thymus and periphery, due to impaired development. However, we found no evidence for defective suppressor activity of miR-155-deficient Tregs, either in vitro or in vivo. Our results indicate that miR-155 contributes to Treg development, but that additional miRNAs control Treg function.

Cerebellar neurodegeneration in the absence of microRNAs

Cerebellar neurodegeneration in the absence of microRNAs

Cerebellar neurodegeneration in the absence of microRNAs

Genome-encoded microRNAs (miRNAs) are potent regulators of gene expression. The significance of miRNAs in various biological processes has been suggested by studies showing an important role of these small RNAs in regulation of cell differentiation. However, the role of miRNAs in regulation of differentiated cell physiology is not well established. Mature neurons express a large number of distinct miRNAs, but the role of miRNAs in postmitotic neurons has not been examined. Here, we provide evidence for an essential role of miRNAs in survival of differentiated neurons. We show that conditional Purkinje cell–specific ablation of the key miRNA-generating enzyme Dicer leads to Purkinje cell death. Deficiency in Dicer is associated with progressive loss of miRNAs, followed by cerebellar degeneration and development of ataxia. The progressive neurodegeneration in the absence of Dicer raises the possibility of an involvement of miRNAs in neurodegenerative disorders.