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In JoVE (1)
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- Ying Yong Sheng Tai Xue Bao = The Journal of Applied Ecology / Zhongguo Sheng Tai Xue Xue Hui, Zhongguo Ke Xue Yuan Shenyang Ying Yong Sheng Tai Yan Jiu Suo Zhu Ban
- American Journal of Human Genetics
- Applied Biochemistry and Biotechnology
- The Biochemical Journal
- Carcinogenesis
- Optics Express
- RNA Biology
- Analytical Biochemistry
- Biochimica Et Biophysica Acta
- American Journal of Physiology. Gastrointestinal and Liver Physiology
- Molecular Pharmacology
- Journal of Cardiovascular Translational Research
- Nucleic Acids Research
- American Journal of Physiology. Gastrointestinal and Liver Physiology
- Waste Management (New York, N.Y.)
- Carcinogenesis
- RNA Biology
- The Journal of Clinical Investigation
- Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
- Blood
- Cell Host & Microbe
- Biochimica Et Biophysica Acta
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Articles by Xiaoxiao Zhang in JoVE
JoVE General
A realização de experimentos de Microarray personalizado MicroRNA
1Department of Pharmacology, University of Minnesota, 2Masonic Cancer Center, University of Minnesota
Um procedimento simples de realizar experimentos de microarray personalizado microRNA é descrito. As etapas incluem isolar RNA, rotulagem RNA e DNA de referência, hibridação as amostras para microarrays, a digitalização da microarrays, quantificar e analisar os sinais de hibridização.
Other articles by Xiaoxiao Zhang on PubMed
[Effects of Neem Seed Extracts on Nitrogen Use Efficiency in Two Different Soils]
Incubation test and pot experiments were conducted with haplic luvisols and hydragric anthrosols to study the effects of neem seed extracts (N I, N II) on nitrification and immobilization of ammonium sulfate. N I could significantly inhibit the nitrification of N applied to the two soils. N II was effective in promoting the immobilization of NH4+(-)N. Pot experiments showed that N II could increase the use efficiency of chemical nitrogen significantly in fimic anthrosols.
High-resolution Mapping of Genotype-phenotype Relationships in Cri Du Chat Syndrome Using Array Comparative Genomic Hybridization
We have used array comparative genomic hybridization to map DNA copy-number changes in 94 patients with cri du chat syndrome who had been carefully evaluated for the presence of the characteristic cry, speech delay, facial dysmorphology, and level of mental retardation (MR). Most subjects had simple deletions involving 5p (67 terminal and 12 interstitial). Genotype-phenotype correlations localized the region associated with the cry to 1.5 Mb in distal 5p15.31, between bacterial artificial chromosomes (BACs) containing markers D5S2054 and D5S676; speech delay to 3.2 Mb in 5p15.32-15.33, between BACs containing D5S417 and D5S635; and the region associated with facial dysmorphology to 2.4 Mb in 5p15.2-15.31, between BACs containing D5S208 and D5S2887. These results overlap and refine those reported in previous publications. MR depended approximately on the 5p deletion size and location, but there were many cases in which the retardation was disproportionately severe, given the 5p deletion. All 15 of these cases, approximately two-thirds of the severely retarded patients, were found to have copy-number aberrations in addition to the 5p deletion. Restriction of consideration to patients with only 5p deletions clarified the effect of such deletions and suggested the presence of three regions, MRI-III, with differing effect on retardation. Deletions including MRI, a 1.2-Mb region overlapping the previously defined cri du chat critical region but not including MRII and MRIII, produced a moderate level of retardation. Deletions restricted to MRII, located just proximal to MRI, produced a milder level of retardation, whereas deletions restricted to the still-more proximal MRIII produced no discernible phenotype. However, MR increased as deletions that included MRI extended progressively into MRII and MRIII, and MR became profound when all three regions were deleted.
A Moderately Thermostable Alkaline Phosphatase from Geobacillus Thermodenitrificans T2: Cloning, Expression and Biochemical Characterization
A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 degrees C, respectively. The enzyme retained a high activity at 45-60 degrees C, while it could be quickly inactivated by a heat treatment at 80 degrees C for 15 min, exhibiting a half-life of 8 min at 70 degrees C. The K(m) and V (max) for pNPP were determined to be 31.5 muM and 430 muM/min at optimal conditions. A divalent cation is essential, with a combination of Mg2+ and Co2+ or Zn2+ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification.
Phosphorylation of Argonaute 2 at Serine-387 Facilitates Its Localization to Processing Bodies
Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.
Differential Expression of MicroRNAs in Early-stage Neoplastic Transformation in the Lungs of F344 Rats Chronically Treated with the Tobacco Carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
While numerous microRNAs (miRNAs) have been reported to alter their expression levels in human lung cancer tissues compared with normal tissues, the function of these miRNAs and their contribution to the long process of lung cancer development remains largely unknown. We applied a tobacco-specific carcinogen-induced cancer model to investigate the involvement of miRNAs in early lung cancer development, which could also provide information on potential, early biomarkers of lung cancers. Male F344 rats were first chronically treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogen present in tobacco products, for up to 20 weeks. The expression profiles of miRNAs in rat lungs were then determined. As measured by miRNA microarrays and confirmed by Northern blot and real-time polymerase chain reaction analyses, NNK treatment reduced the expression of a number of miRNAs, such as miR-101, miR-126*, miR-199 and miR-34. Significantly, these miRNAs overlap with previously published reports on altered miRNA expression in human lung cancer samples. These miRNAs might, therefore, represent early-response miRNAs that signify the molecular changes associated with pulmonary tumorigenesis. Moreover, we identified cytochrome P450 (CYP) 2A3, a critical enzyme in rat lungs that activates NNK to render it carcinogenic, as a potential target of miR-126*. NNK treatment in rats repressed miR-126* but induced CYP2A3 expression, a mechanism that may potentiate the oncogenic effects of NNK.
Optimizing Distance Image Quality of an Aspheric Multifocal Intraocular Lens Using a Comprehensive Statistical Design Approach
The AcrySof ReSTOR intraocular lens (IOL) is a multifocal lens with state-of-the-art apodized diffractive technology, and is indicated for visual correction of aphakia secondary to removal of cataractous lenses in adult patients with/without presbyopia, who desire near, intermediate, and distance vision with increased spectacle independence. The multifocal design results in some optical contrast reduction, which may be improved by reducing spherical aberration. A novel patent-pending approach was undertaken to investigate the optical performance of aspheric lens designs. Simulated eyes using human normal distributions were corrected with different lens designs in a Monte Carlo simulation that allowed for variability in multiple surgical parameters (e.g. positioning error, biometric variation). Monte Carlo optimized results indicated that a lens spherical aberration of -0.10 microm provided optimal distance image quality.
MicroRNAs Identified in Highly Purified Liver-derived Mitochondria May Play a Role in Apoptosis
MicroRNAs (miRNAs) are a class of small approximately 22 nt noncoding (nc) RNAs that regulate gene expression post-transcriptionally by direct binding to target sites on mRNAs. They comprise more than 1,000 novel species in mammalian cells and exert their function by modulating gene expression through several different mechanisms, including translational inhibition, and/or degradation of target mRNAs. Mitochondria maintain and express their own genome, which is distinct from the nuclear transcriptional and translational apparatus. Thus, they provide a potential site for miRNA mediated post-transcriptional regulation. To determine whether they maintain a unique miRNA population, we examined the miRNA profile from highly purified and RNase treated mitochondria from adult rat liver. Fifteen miRNAs were identified by microarray analysis of which, five were confirmed by TaqMan 5'nuclease assays using rat specific probes. Functional analysis of the miRNAs indicated that they were not targeted to the mitochondrial genome nor were they complementary to nuclear RNAs encoding mitochondrial proteins. Rather, the mitochondria-associated miRNAs appear to be involved in the expression of genes associated with apoptosis, cell proliferation, and differentiation. Given the central role that mitochondria play in apoptosis, the results suggest that they might serve as reservoirs of select miRNAs that may modulate these processes in a coordinate fashion.
Stripping Custom MicroRNA Microarrays and the Lessons Learned About Probe-slide Interactions
Microarrays have been used extensively in gene expression profiling and genotyping studies. To reduce the high cost and enhance the consistency of microarray experiments, it is often desirable to strip and reuse microarray slides. Our genome-wide analysis of microRNA expression involves the hybridization of fluorescently labeled nucleic acids to custom-made, spotted DNA microarrays based on GAPSII-coated slides. We describe here a simple and effective method to regenerate such custom microarrays that uses a very low-salt buffer to remove labeled nucleic acids from microarrays. Slides can be stripped and reused multiple times without significantly compromising data quality. Moreover, our analyses of the performance of regenerated slides identifies parameters that influence the attachment of oligonucleotide probes to GAPSII slides, shedding light on the interactions between DNA and the microarray surface and suggesting ways in which to improve the design of oligonucleotide probes.
Stable Argonaute2 Overexpression Differentially Regulates MicroRNA Production
microRNAs are a class of small RNA molecules that associate with Argonaute proteins to regulate gene expression. Argonaute proteins not only mediate the functions of microRNAs, but also play an essential role in the biogenesis of microRNAs. Here, we report that stable, long-term overexpression of Argonaute2, an Argonaute isoform, induces the production of a number of microRNAs, such as the let-7 family of microRNAs, in 293T cells. On the other hand, the expression of many microRNAs is insensitive to elevated Argonaute levels, and microRNAs in the miR-17-92 and homologous clusters are even down-regulated. The down-regulation may result from the let-7-mediated inhibition of the expression of Myc, which positively controls the transcription of the microRNAs clusters. Our data suggest that human cells have a mechanism for microRNA homeostasis, and that overexpression of a general microRNA processing factor can differentially regulate the expression of specific microRNAs.
Polysome Trafficking of Transcripts and MicroRNAs in Regenerating Liver After Partial Hepatectomy
Liver regeneration after 70% partial hepatectomy (PH) in rats induces >95% of hepatocytes to undergo two rounds of semisynchronous cell replication. Gene expression is controlled primarily by posttranscriptional processing, including changes in mRNA stability. However, the translational activity of a specific mRNA can also be modulated after PH, resulting in significant uncoupling of protein and transcript levels relative to quiescent liver for many genes including c-myc and p53. Although the precise mechanism by which this uncoupling occurs is unknown, the polysomal association of mRNA and microRNA (miRNA) can significantly modulate rate of decay as well as translational activity. Thus we characterized the association of c-myc and p53 mRNAs and miRNAs in free and cytoskeleton- and membrane-bound polysome populations 3, 6, and 24 h after PH. The transcripts for c-myc and p53 were differentially distributed in the three discrete polysome populations, and this was dramatically modulated during liver regeneration. Nascent polysome-associated p53 and c-myc proteins were also differentially expressed in the free and cytoskeleton- and membrane-bound polysomes and significantly uncoupled from transcript levels relative to nonresected liver. At least 85 miRNAs were associated with the three polysome populations, and their abundance and distribution changed significantly during liver regeneration. These data suggest that posttranscriptional control of c-myc and p53 protein expression is associated with the translocation of transcripts between the different polyribosomes. The alteration of expression for the same transcript in different polysome populations may, in part, be due to the action of miRNAs.
Mu-Opioid Receptor Agonists Differentially Regulate the Expression of MiR-190 and NeuroD
The agonists of mu-opioid receptor (OPRM1) induce extracellular signal-regulated kinase (ERK) phosphorylation through different pathways: morphine uses the protein kinase C (PKC)-pathway, whereas fentanyl functions in a beta-arrestin2-dependent manner. In addition, the two pathways result in the different cellular location of phosphorylated ERK and the activation of different sets of transcriptional factors. In the current study, the influence of the two pathways on the expression of microRNAs (miRNAs) was investigated. After treating the primary culture of rat hippocampal neurons and the mouse hippocampi with morphine or fentanyl for 3 days, seven miRNAs regulated by one or two of the agonists were identified. One of the identified miRNAs, miR-190, was down-regulated by fentanyl but not by morphine. This down-regulation was attenuated by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126), which blocks the phosphorylation of ERK. When fentanyl-induced but not morphine-induced ERK phosphorylation was blocked in the primary cultures from beta-arrestin2(-/-) mouse, fentanyl did not decrease the expression of miR-190. However, a PKC inhibitor that blocked morphine-induced ERK phosphorylation specifically had no effect on the miR-190 down-regulation. Therefore the decrease in miR-190 expression resulted from the agonist-selective ERK phosphorylation. In addition, the expressional changes in one of the miR-190 targets, neurogenic differentiation 1 (NeuroD), correlated with those in miR-190 expression, suggesting the OPRM1 could regulate the NeuroD pathways via the control of miR-190 expression.
Regulation of Mammalian MicroRNA Expression
MicroRNAs are widely expressed, approximately 22-nucleotide-long non-protein-coding RNAs. They are first transcribed as much longer primary transcripts, which then undergo a series of processing steps to yield the single-stranded, mature microRNAs. Like other classes of RNAs in a cell, the biogenesis of microRNAs is under the control of various internal and external cues. This review summaries our current knowledge of how the expression of mammalian microRNAs is regulated transcriptionally and post-transcriptionally.
The Terminal Loop Region Controls MicroRNA Processing by Drosha and Dicer
microRNAs are widely expressed, ∼22-nt-long regulatory RNAs. They are first transcribed as much longer primary transcripts, which then undergo a series of processing steps to yield the single-stranded, mature microRNAs, although the mechanisms are incompletely understood. Here, we show that the terminal loop region of human primary microRNA transcripts is an important determinant of microRNA biogenesis. Mutations that restrain the terminal loop region inhibit Drosha processing of primary microRNA transcripts as well as Dicer processing of precursor microRNA transcripts in vitro. The inhibition may result from lower enzyme turnover on the mutant transcripts. Consequently, the mutations reduce miRNA maturation in transfected human cells. We conclude that a flexible terminal loop region is critical for microRNA processing.
Identification of MicroRNAs During Rat Liver Regeneration After Partial Hepatectomy and Modulation by Ursodeoxycholic Acid
New gene regulation study tools such as microRNA (miRNA or miR) analysis may provide unique insights into the remarkable ability of the liver to regenerate. In addition, we have previously shown that ursodeoxycholic acid (UDCA) modulates mRNA levels during liver regeneration. Bile acids are also homeotrophic sensors of functional hepatic capacity. The present study was designed to determine whether miRNAs are modulated in rats following 70% partial hepatectomy (PH) and elucidate the role of UDCA in regulating miRNA expression during liver regeneration (LR). Total RNA was isolated from livers harvested at 3-72 h following 70% PH or sham operations, from both 0.4% (wt/wt) UDCA and control diet-fed animals. By using a custom microarray platform we found that several miRNAs are significantly altered after PH by >1.5-fold, including some previously described as modulators of cell proliferation, differentiation, and death. In particular, expression of miR-21 was increased after PH. Functional modulation of miR-21 in primary rat hepatocytes increased cell proliferation and viability. Importantly, UDCA was a strong inducer of miR-21 both during LR and in cultured HepG2 cells. In fact, UDCA feeding appeared to induce a sustained increase of proliferative miRNAs observed at early time points after PH. In conclusion, miRNAs, in particular miR-21, may play a significant role in modulating proliferation and cell cycle progression genes after PH. miR-21 is additionally induced by UDCA in both regenerating rat liver and in vitro, which may represent a new mechanism behind UDCA biological functions.
Environmental Friendly Leaching Reagent for Cobalt and Lithium Recovery from Spent Lithium-ion Batteries
We investigated an environmentally friendly leaching process for the recovery of cobalt and lithium from the cathode active materials of spent lithium-ion batteries. The easily degradable organic acid DL-malic acid (C(4)H(5)O(6)) was used as a leaching reagent. The structural, morphology of the cathode materials before and after leaching were characterized by X-ray diffraction (XRD) and scanning electronic microscopy (SEM). The amount of Co and Li present in the leachate was determined by atomic absorption spectrophotometry (AAS). Conditions for achieving a recovery of more than 90 wt.% Co and nearly 100 wt.% Li were determined experimentally by varying the concentrations of leachant, time and temperature of the reaction as well as the initial solid-to-liquid ratio. We found that hydrogen peroxide in a DL-malic acid solution is an effective reducing agent because it enhances the leaching efficiency. Leaching with 1.5M DL-malic acid, 2.0 vol.% hydrogen peroxide and a S:L of 20 g L(-1) in a batch extractor results in a highly efficient recovery of the metals within 40 min at 90 °C.
Alteration of MicroRNA Expression in Vinyl Carbamate-induced Mouse Lung Tumors and Modulation by the Chemopreventive Agent Indole-3-carbinol
MicroRNAs (miRNAs) are small, non-protein-coding RNAs that can function as tumor suppressors or oncogenes. Deregulation of miRNA expression has been reported in lung cancer. However, modulation of miRNA expression by chemopreventive agents remains to be defined. In the present study, we examined if the chemopreventive agent indole-3-carbinol (I3C) reversed vinyl carbamate (VC)-induced deregulation of miRNA levels in lung tissues of female A/J mice. Lung tissues were obtained from a previous chemoprevention study, in which mice were treated with VC and given I3C in the diet for 15 weeks. Microarray studies revealed alterations in the expression of a number of miRNAs in lung tumors relative to that of normal lungs. miR-21, mir-31, miR-130a, miR-146b and miR-377 were consistently upregulated, whereas miR-1 and miR-143 were downregulated in lung tumors relative to normal lungs. In mice treated with VC and given I3C in the diet, levels of miR-21, mir-31, miR-130a, miR-146b and miR-377 were reduced relative to the level in mice treated with the carcinogen only. The results of the microarray study were confirmed by quantitative reverse transcription-polymerase chain reaction and gel analysis of polymerase chain reaction products. Further studies with miR-21 indicated that phosphatase and tensin homolog, programmed cell death 4 and rich protein with Kazal motifs are potential targets for the oncogenic effect of miR-21 and the chemopreventive activity of I3C. Taken together, we showed here that miRNAs are deregulated during VC-induced mouse lung tumorigenesis and their levels are modulated by I3C. Therefore, miRNAs and their target genes are promising biomarkers for the diagnosis of lung cancer and efficacy of chemopreventive/chemotherapeutic agents.
Mature MicroRNAs Identified in Highly Purified Nuclei from HCT116 Colon Cancer Cells
MicroRNAs (miRNAs) have emerged as one of the major regulatory mechanisms of gene expression. A major function of miRNAs involves the post-transcriptional regulation of target mRNAs, which is reported to occur primarily in the cytoplasm. However, there is a significant amount of evidence demonstrating the existence of small non-coding RNAs, including small-interfering RNA (siRNA), miRNA, and Piwi-interacting RNA (piRNA) in the nucleus. In order to elucidate the potential subcellular localizations and functions of miRNAs, we have identified numerous miRNAs that are present in isolated nuclei from human colon cancer HCT116 cells. MicroRNA profiles were compared between cytoplasmic and nuclear fractions of the HCT116 cell line on the basis of multiple microarray analyses. MicroRNA species showing significant existence in isolated and highly purified populations of nuclei were selected and further tested with RT-PCR. The nuclear localization of the mature form of miRNAs was verified again by control RT-PCR excluding the detection of premature forms of miRNA, such as pri-miRNA or pre-miRNA. The elevated levels of representative miRNAs identified in purified nuclei were confirmed by Northern blot analysis, supporting the notion that significant numbers of mature miRNAs exist not only in the cytoplasm but also in the nucleus. These results will likely provide a basis for further studies concerning the intracellular trafficking and nuclear location of miRNAs.
Hypoxia-induced MicroRNA-424 Expression in Human Endothelial Cells Regulates HIF-α Isoforms and Promotes Angiogenesis
Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.
Rapid Monodisperse Microencapsulation of Single Cells
A microfluidic device was designed having the ability to continuously produce monodisperse microcapsules with controlled cell loading. The design included stages of inertial focusing, droplet generation, and photopolymerization. Prototype microfluidic devices were fabricated in polydimethylsiloxane (PDMS) to demonstrate each stage using poly(ethylene-glycol)-diacrylate (PEGDA) as the encapsulating material and oil as the droplet-containing medium, creating a water-in-oil emulsion. 10.3-µm-diameter fluorescent polystyrene beads were used as cell simulants. In the first stage, inertial focusing was demonstrated using a straight-channel configuration. In the second stage, droplets with a 60±5µm diameter were generated. In the third stage, successful encapsulation of the beads in hydrogel droplets was verified. This technology can significantly impact a wide research area ranging from cellular therapeutics to single-cell manipulation.
MiR-221 and MiR-155 Regulate Human Dendritic Cell Development, Apoptosis, and IL-12 Production Through Targeting of P27kip1, KPC1, and SOCS-1
Dendritic cells (DCs) are potent antigen-presenting cells derived from hematopoietic progenitor cells and circulating monocytes. To investigate the role of microRNAs (miRNAs) during DC differentiation, maturation, and function, we profiled miRNA expression in human monocytes, immature DCs (imDCs), and mature DCs (mDCs). Stage-specific, differential expression of 27 miRNAs was found during monocyte differentiation into imDCs and mDCs. Among them, decreased miR-221 and increased miR-155 expression correlated with p27(kip1) accumulation in DCs. Silencing of miR-221 or overexpressing of miR-155 in DCs resulted in p27(kip1) protein increase and DC apoptosis. Moreover, mDCs from miR-155(-/-) mice were less apoptotic than those from wild-type mice. Silencing of miR-155 expression had little effect on DC maturation but reduced IL-12p70 production, whereas miR-155 overexpression in mDCs enhanced IL-12p70 production. Kip1 ubiquitination-promoting complex 1, suppressor of cytokine signaling 1, and CD115 (M-CSFR) were functional targets of miR-155. Furthermore, we provide evidence that miR-155 indirectly regulated p27(kip1) protein level by targeting Kip1 ubiquitination-promoting complex 1. Thus, our study uncovered miRNA signatures during monocyte differentiation into DCs and the new regulatory role of miR-221 and miR-155 in DC apoptosis and IL-12p70 production.
Structural and Functional Analysis of a Plant Resistance Protein TIR Domain Reveals Interfaces for Self-association, Signaling, and Autoregulation
The Toll/interleukin-1 receptor (TIR) domain occurs in animal and plant immune receptors. In the animal Toll-like receptors, homodimerization of the intracellular TIR domain is required for initiation of signaling cascades leading to innate immunity. By contrast, the role of the TIR domain in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant immune resistance proteins is poorly understood. L6 is a TIR-NB-LRR resistance protein from flax (Linum usitatissimum) that confers resistance to the flax rust phytopathogenic fungus (Melampsora lini). We determine the crystal structure of the L6 TIR domain and show that, although dispensable for pathogenic effector protein recognition, the TIR domain alone is both necessary and sufficient for L6 immune signaling. We demonstrate that the L6 TIR domain self-associates, most likely forming a homodimer. Analysis of the structure combined with site-directed mutagenesis suggests that self-association is a requirement for immune signaling and reveals distinct surface regions involved in self-association, signaling, and autoregulation.
Drosha Processing Controls the Specificity and Efficiency of Global MicroRNA Expression
microRNAs (miRNAs) are a large family of approximately 22-nucleotide-long RNAs that regulate gene expression. They are first transcribed as long, primary transcripts, which then undergo a series of processing steps to generate the single-stranded, mature miRNAs. Here, we showed that Drosha cleaved hundreds of human primary miRNA transcript substrates with different efficiencies in vitro. The differential Drosha susceptibility of the primary miRNA transcripts significantly correlated with the expression of the corresponding, mature miRNAs in vivo. Conserved miRNAs were more efficiently expressed in vivo, and their primary transcripts were also better Drosha substrates in vitro. Combining secondary structure prediction and statistical analyses, we identified features in human primary miRNA transcripts that predisposed miRNAs to efficient Drosha processing in vitro as well as to better expression in vivo. We propose that the selectivity of Drosha action contributes greatly to the specificity and efficiency of miRNA biogenesis. Moreover, this study serves as an example of substrate specificity of a biochemical reaction regulating gene expression at a global scale in vivo. This article is part of a Special Issue entitled: MicroRNA's in viral gene regulation.
Up-regulated Expression of Ezrin and C-Met Proteins Are Related to the Metastasis and Prognosis of Gastric Carcinomas
Recent publications demonstrated that abnormal expression of Ezrin and c-Met proteins were related to carcinogenesis, metastasis and prognosis of various sorts of tumors. In this study we detected the expressions of Ezrin and c-Met proteins in normal gastric mucosa, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma and analyzed the correlations with metastasis and prognosis of gastric carcinomas. The results demonstrated that both Ezrin and c-Met overexpression were related to the occurrence and progression of gastric carcinoma. Our findings also demonstrated that combined detection of these two tumor-specific biomarkers in gastric carcinomas can provide additional efficacy in predicting the patients' outcomes.
