Genome-wide profiling identified a set of miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells

A major challenge in cancer research field is to define molecular features that distinguish cancer stem cells from normal stem cells. In this study, we compared microRNA (miRNA) expression profiles in human glioblastoma stem cells and normal neural stem cells using combined microarray and deep sequencing analyses. These studies allowed us to identify a set of 10 miRNAs that are considerably up-regulated or down-regulated in glioblastoma stem cells. Among them, 5 miRNAs were further confirmed to have altered expression in three independent lines of glioblastoma stem cells by real-time RT-PCR analysis. Moreover, two of the miRNAs with increased expression in glioblastoma stem cells also exhibited elevated expression in glioblastoma patient tissues examined, while two miRNAs with decreased expression in glioblastoma stem cells displayed reduced expression in tumor tissues. Furthermore, we identified two oncogenes, NRAS and PIM3, as downstream targets of miR-124, one of the down-regulated miRNAs; and a tumor suppressor, CSMD1, as a downstream target of miR-10a and miR-10b, two of the up-regulated miRNAs. In summary, this study led to the identification of a set of miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells. Characterizing the role of these miRNAs in glioblastoma stem cells may lead to the development of miRNA-based therapies that specifically target tumor stem cells, but spare normal stem cells.     

三唑磷、氟虫腈及其复配剂对四种miRNAs在斑马鱼组织中表达的影响

MicroRNAs(miRNAs)是一类长约22 nt的非编码小RNA,在基因表达中起重要调控作用.已有研究表明,农药三唑磷和氟虫腈能影响斑马鱼全鱼组织中部分miRNAs的正常表达,但未见对miRNA表达的组织特异性的研究.本研究采用荧光定量PCR技术研究了经三唑磷微乳剂、氟虫腈微乳剂及其复配剂处理后,四种miRNA(...     

Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes

MicroRNAs (miRNAs) play key roles in gene expression regulation by guiding Argonaute (AGO)-containing microribonucleoprotein (miRNP) effector complexes to target polynucleotides. There are still uncertainties about how miRNAs interact with mRNAs. Here we employed a biochemical approach to isolate AGO-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with a previously described anti-AGO antibody. Co-immunoprecipitated (co-IPed) RNAs were subjected to downstream Affymetrix Human Gene 1.0 ST microarray analysis. During rigorous validation, the “RIP-Chip” assay identified target mRNAs specifically associated with AGO complexes. RIP-Chip was performed after transfecting brain-enriched miRNAs (miR-107, miR-124, miR-128, and miR-320) and nonphysiologic control miRNA to identify miRNA targets. As expected, the miRNA transfections altered the mRNA content of the miRNPs. Specific mRNA species recruited to miRNPs after miRNA transfections were moderately in agreement with computational target predictions. In addition to recruiting mRNA targets into miRNPs, miR-107 and to a lesser extent miR-128, but not miR-124 or miR-320, caused apparent exclusion of some mRNAs that are normally associated with miRNPs. MiR-107 and miR-128 transfections also result in decreased AGO mRNA and protein levels. However, AGO mRNAs were not recruited to miRNPs after either miR-107 or miR-128 transfection, confirming that miRNAs may alter gene expression without stable association between particular mRNAs and miRNPs. In summary, RIP-Chip assays constitute an optimized, validated, direct, and high-throughput biochemical assay that provides data about specific miRNA:mRNA interactions, as well as global patterns of regulation by miRNAs.     

Targeting miR-9 in Glioma Stem Cell-Derived Extracellular Vesicles: A Novel Diagnostic and Therapeutic Biomarker

BackgroundGlioblastoma (GBM) is a lethal brain tumor with no effective strategies in early diagnosis and treatment. This study was aimed to assess the miRNA expression profiles in EVs from CSF and tissue of glioblastoma patients to identify significantly upregulated miRNAs and investigate the underlying neoplastic mechanisms.MethodsEVs were measured by TEM and NTA assays. Differentially regulated miRNAs were measured using RNA sequencing in GBM CSF EVs and in GBM tissues compared with controls. RT-qPCR was employed to analyze miRNA and gene expression. Luciferase report assay was used to investigate gene target of miR-9. The proliferation ability was detected by EdU and CCK-8 experiment while cell migration was measured by transwell and wound healing assay.ResultsThe expression level of miR-9 was significantly higher in GBM CSF EVs and tissues than controls (p = 0.038). The area under curve for CSF EV miR-9 was 0.800 (95% CI: 0.583–1.000, p = 0.033). The expression of miR-9 was significantly higher in Glioma stem cells (GSCs) and GSC-derived EVs than in glioblastoma cells. GSC-derives EVs could promote GBM growth and migration Moreover, inhibition of miR-9 in GSCs showed the reverse anti-tumor effects through secreted EVs. MiR-9 could bind to the 3’UTR region of DACT3 and suppress its expression. The miR-9/DACT3 axis might attribute to GBM malignant phenotype.ConclusionMiR-9 in CSF EVs may act as a novel diagnostic biomarker for GBM and targeting miR-9 by GSC-derived EVs may be a specific and efficient strategy for GBM biotherapy.     

A specific miRNA signature in the peripheral blood of glioblastoma patients

The prognosis of patients afflicted by glioblastoma remains poor. Biomarkers for the disease would be desirable in order to allow for an early detection of tumor progression or to indicate rapidly growing tumor subtypes requiring more intensive therapy. In this study, we investigated whether a blood-derived specific miRNA fingerprint can be defined in patients with glioblastoma. To this end, miRNA profiles from the blood of 20 patients with glioblastoma and 20 age- and sex-matched healthy controls were compared. Of 1158 tested miRNAs, 52 were significantly deregulated, as assessed by unadjusted Student's t-test at an alpha level of 0.05. Of these, two candidates, miR-128 (up-regulated) and miR-342-3p (down-regulated), remained significant after correcting for multiple testing by Benjamini-Hochberg adjustment with a p-value of 0.025. The altered expression of these two biomarkers was confirmed in a second cohort of glioblastoma patients and healthy controls by real-time PCR and validated for patients who had received neither radio- nor chemotherapy and for patients who had their glioblastomas resected more than 6 months ago. Moreover, using machine learning, a comprehensive miRNA signature was obtained that allowed for the discrimination between blood samples of glioblastoma patients and healthy controls with an accuracy of 81% [95% confidence interval (CI) 78-84%], specificity of 79% (95% CI 75-83%) and sensitivity of 83% (95% CI 71-85%). In summary, our proof-of-concept study demonstrates that blood-derived glioblastoma-associated characteristic miRNA fingerprints may be suitable biomarkers and warrant further exploration.     

Genome-wide analysis of aberrantly expressed circulating miRNAs in patients with coal workers’ pneumoconiosis

Emerging evidence indicates that microRNAs (miRNAs), a class of small non-coding regulatory RNAs, have important roles in multiple biological processes. To determine the potential contribution of miRNAs to coal workers’ pneumoconiosis (CWP), we comprehensively surveyed and identified differentially expressed miRNA profiles in patients with CWP by small RNA sequencing and analysis. Mixed serum samples from the different stages of CWP and the control samples were subjected to deep sequencing by applying next-generation sequencing technology. Samples at different disease stages exhibited inconsistent miRNA expression profiles and differentially expressed miRNA profiles. Generally, these miRNAs were dynamically expressed across the different disease stages and showed various relative expression levels. Some miRNAs (such as miR-18a*, 149, 222 and 671-3p) were consistently up-regulated or down-regulated in the different stages of CWP samples. Most of the aberrantly expressed miRNAs showed a down-regulation trend. Differentially expressed miRNAs were also subjected to pairwise comparison between the different stages. Some miRNAs showed significant inconsistent expression trends across the three stages, although they were not significantly dysregulated based on the control sample. Furthermore, a series of special miRNAs organized into miRNA gene clusters and gene families were also surveyed for aberrant expression (such as mir-200 gene family and mir-222 gene cluster). According to experimentally validated target mRNAs of the aberrantly and abundantly expressed miRNAs, functional enrichment analysis suggests that these miRNAs play important roles in various biological processes, including lung tumorigenesis. In summary, we demonstrated that aberrantly expressed circulating miRNAs showed dynamic expression patterns across diseased samples, which suggests that these miRNAs may have critical roles in the occurrence and development of CWP. In addition, some significantly dysregulated miRNAs may be potential non-invasive diagnosis biomarkers based on further study.     

Hepato-specific microRNA-122 facilitates accumulation of newly synthesized miRNA through regulating PRKRA

microRNAs (miRNAs) are a versatile class of non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. In this study, we employed 3'-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNAs demonstrated a specific enrichment of several known miR-122 targets such as CAT-1 (also called SLC7A1), ADAM17 and BCL-w. Using microarray analysis of affinity purified RNAs, we also discovered many candidate target genes of miR-122. Among these candidates, we confirmed that protein kinase, interferon-inducible double-stranded RNA-dependent activator (PRKRA), a Dicer-interacting protein, is a direct target gene of miR-122. miRNA quantitative-RT-PCR results indicated that miR-122 and small interfering RNA against PRKRA may facilitate the accumulation of newly synthesized miRNAs but did not detectably affect endogenous miRNAs levels. Our findings will lead to further understanding of multiple functions of this hepato-specific miRNA. We conclude that miR-122 could repress PRKRA expression and facilitate accumulation of newly synthesized miRNAs.     

microRNA-Seq reveals cocaine-regulated expression of striatal microRNAs

MicroRNAs (miRNAs) are small RNAs that modulate gene expression by binding target mRNAs. The hundreds of miRNAs expressed in the brain are critical for synaptic development and plasticity. Drugs of abuse cause lasting changes in the limbic regions of the brain that process reward, and addiction is viewed as a form of aberrant neuroplasticity. Using next-generation sequencing, we cataloged miRNA expression in the nucleus accumbens and at striatal synapses in control and chronically cocaine-treated mice. We identified cocaine-responsive miRNAs, synaptically enriched and depleted miRNA families, and confirmed cocaine-induced changes in protein expression for several predicted synaptic target genes. The miR-8 family, known for its roles in cancer, is highly enriched and cocaine regulated at striatal synapses, where its members may affect expression of cell adhesion molecules. Synaptically enriched cocaine-regulated miRNAs may contribute to long-lasting drug-induced plasticity through fine-tuning regulatory pathways that modulate the actin cytoskeleton, neurotransmitter metabolism, and peptide hormone processing.     

Identification of mouse serum miRNA endogenous references by global gene expression profiles

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments.     

MiR-107 and MiR-185 Can Induce Cell Cycle Arrest in Human Non Small Cell Lung Cancer Cell Lines

Background

MicroRNAs (miRNAs) are short single stranded noncoding RNAs that suppress gene expression through either translational repression or degradation of target mRNAs. The annealing between messenger RNAs and 5′ seed region of miRNAs is believed to be essential for the specific suppression of target gene expression. One miRNA can have several hundred different targets in a cell. Rapidly accumulating evidence suggests that many miRNAs are involved in cell cycle regulation and consequentially play critical roles in carcinogenesis.

Methodology/Principal Findings

Introduction of synthetic miR-107 or miR-185 suppressed growth of the human non-small cell lung cancer cell lines. Flow cytometry analysis revealed these miRNAs induce a G1 cell cycle arrest in H1299 cells and the suppression of cell cycle progression is stronger than that by Let-7 miRNA. By the gene expression analyses with oligonucleotide microarrays, we find hundreds of genes are affected by transfection of these miRNAs. Using miRNA-target prediction analyses and the array data, we listed up a set of likely targets of miR-107 and miR-185 for G1 cell cycle arrest and validate a subset of them using real-time RT-PCR and immunoblotting for CDK6.

Conclusions/Significance

We identified new cell cycle regulating miRNAs, miR-107 and miR-185, localized in frequently altered chromosomal regions in human lung cancers. Especially for miR-107, a large number of down-regulated genes are annotated with the gene ontology term ‘cell cycle’. Our results suggest that these miRNAs may contribute to regulate cell cycle in human malignant tumors.     

Novel Anti-Apoptotic MicroRNAs 582-5p and 363 Promote Human Glioblastoma Stem Cell Survival via Direct Inhibition of Caspase 3, Caspase 9, and Bim

Glioblastoma is the most common and lethal primary brain tumor. Tumor initiation and recurrence are likely caused by a sub-population of glioblastoma stem cells, which may derive from mutated neural stem and precursor cells. Since CD133 is a stem cell marker for both normal brain and glioblastoma, and to better understand glioblastoma formation and recurrence, we looked for dys-regulated microRNAs in human CD133+ glioblastoma stem cells as opposed to CD133+ neural stem cells isolated from normal human brain. Using FACS sorting of low-passage cell samples followed by microRNA microarray analysis, we found 43 microRNAs that were dys-regulated in common in three separate CD133+ human glioblastomas compared to CD133+ normal neural stem cells. Among these were several microRNAs not previously associated with cancer. We then verified the microRNAs dys-regulated in glioblastoma using quantitative real time PCR and Taqman analysis of the original samples, as well as human GBM stem cell and established cell lines and many human specimens. We show that two candidate oncogenic microRNAs, miR-363 and miR-582-5p, can positively influence glioblastoma survival, as shown by forced expression of the microRNAs and their inhibitors followed by cell number assay, Caspase 3/7 assay, Annexin V apoptosis/fluorescence activated cell sorting, siRNA rescue of microRNA inhibitor treatment, as well as 3′UTR mutagenesis to show luciferase reporter rescue of the most successful targets. miR-582-5p and miR-363 are shown to directly target Caspase 3, Caspase 9, and Bim.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1

MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.