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1.  Nonviral Direct Conversion of Primary Mouse Embryonic Fibroblasts to Neuronal Cells 
Transdifferentiation, where differentiated cells are reprogrammed into another lineage without going through an intermediate proliferative stem cell-like stage, is the next frontier of regenerative medicine. Wernig et al. first described the direct conversion of fibroblasts into functional induced neuronal cells (iNs). Subsequent reports of transdifferentiation into clinically relevant neuronal subtypes have further endorsed the prospect of autologous cell therapy for neurodegenerative disorders. So far, all published neuronal transdifferentiation protocols rely on lentiviruses, which likely precludes their clinical translation. Instead, we delivered plasmids encoding neuronal transcription factors (Brn2, Ascl1, Myt1l) to primary mouse embryonic fibroblasts with a bioreducible linear poly(amido amine). The low toxicity and high transfection efficiency of this gene carrier allowed repeated dosing to sustain high transgene expression levels. Serial 0.5 µg cm−2 doses of reprogramming factors delivered at 48-hour intervals produced up to 7.6% Tuj1+ (neuron-specific class III β-tubulin) cells, a subset of which expressed MAP2 (microtubule-associated protein 2), tau, and synaptophysin. A synapsin-red fluorescent protein (RFP) reporter helped to identify more mature, electrophysiologically active cells, with 24/26 patch-clamped RFP+ cells firing action potentials. Some non-virally induced neuronal cells (NiNs) were observed firing multiple and spontaneous action potentials. This study demonstrates the feasibility of nonviral neuronal transdifferentiation, and may be amenable to other transdifferentiation processes.
doi:10.1038/mtna.2012.25
PMCID: PMC3411320  PMID: 23344148
2.  MicroRNA in pluripotent stem cells 
Regenerative medicine  2010;5(4):545-555.
SUMMARY
Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are characterized by their ability to self renew and differentiate into any cell type. The molecular mechanism behind this process is a complex interplay between the transcriptional factors with epigenetic regulators and signaling pathways. MicroRNAs are an integral part of this regulatory network with essential roles in pluripotent maintenance, proliferation and differentiation. MicroRNAs are a class of small non-coding RNAs that target protein-encoding mRNA to inhibit translation and protein synthesis. Discovered close to two decades ago, microRNAs have rapidly emerged as key regulatory molecules in several critical cellular processes across species. Recent studies have begun to clarify the specific role of microRNA in regulatory circuitries that control self renewal and pluripotency of both ESC and iPSC. These advances suggest a critical role for microRNAs in the process of reprogramming of somatic cells to pluripotent cells.
doi:10.2217/rme.10.34
PMCID: PMC3077715  PMID: 20632858
microRNA; epigenetics; embryonic stem cells; induced pluripotent stem cells; Review
3.  An Integrative Analysis of microRNA and mRNA Expression—A Case Study 
Cancer Informatics  2008;6:369-379.
Background
MicroRNAs are believed to play an important role in gene expression regulation. They have been shown to be involved in cell cycle regulation and cancer. MicroRNA expression profiling became available owing to recent technology advancement. In some studies, both microRNA expression and mRNA expression are measured, which allows an integrated analysis of microRNA and mRNA expression.
Results
We demonstrated three aspects of an integrated analysis of microRNA and mRNA expression, through a case study of human cancer data. We showed that (1) microRNA expression efficiently sorts tumors from normal tissues regardless of tumor type, while gene expression does not; (2) many microRNAs are down-regulated in tumors and these microRNAs can be clustered in two ways: microRNAs similarly affected by cancer and microRNAs similarly interacting with genes; (3) taking let-7f as an example, targets genes can be identified and they can be clustered based on their relationship with let-7f expression.
Discussion
Our findings in this paper were made using novel applications of existing statistical methods: hierarchical clustering was applied with a new distance measure—the co-clustering frequency—to identify sample clusters that are stable; microRNA-gene correlation profiles were subject to hierarchical clustering to identify microRNAs that similarly interact with genes and hence are likely functionally related; the clustering of regression models method was applied to identify microRNAs similarly related to cancer while adjusting for tissue type and genes similarly related to microRNA while adjusting for disease status. These analytic methods are applicable to interrogate multiple types of -omics data in general.
PMCID: PMC2623315  PMID: 19259417
clustering; expression; microarray; microRNA
4.  MicroRNAs: Meta-controllers of gene expression in synaptic activity emerge as genetic and diagnostic markers of human disease 
Pharmacology & therapeutics  2011;130(1):26-37.
MicroRNAs are members of the non-protein-coding family of RNAs. They serve as regulators of gene expression by modulating the translation and/or stability of messenger RNA targets. The discovery of microRNAs has revolutionized the field of cell biology, and has permanently altered the prevailing view of a linear relationship between gene and protein expression. The increased complexity of gene regulation is both exciting and daunting, as emerging evidence supports a pervasive role for microRNAs in virtually every cellular process. This review briefly describes microRNA processing and formation of RNA-induced silencing complexes, with a focus on the role of RNA binding proteins in this process. We also discuss mechanisms for microRNA-mediated regulation of translation, particularly in dendritic spine formation and function, and the role of microRNAs in synaptic plasticity. We then discuss the evidence for altered microRNA function in cognitive brain disorders, and the effect of gene mutations revealed by single nucleotide polymorphism analysis on altered microRNA function and human disease. Further, we present evidence that altered microRNA expression in circulating fluids such as plasma/serum can correlate with, and serve as, novel diagnostic biomarkers of human disease.
doi:10.1016/j.pharmthera.2011.01.004
PMCID: PMC3043141  PMID: 21256154
MicroRNA; 3′ untranslated region; brain disorders; dendrite; diagnostic marker; eukaryotic initiation factor; fragile X mental retardation protein; human disease; mRNA stability; plasticity; RNA binding protein; RNA-induced silencing complex; single nucleotide polymorphism; translation
5.  MicroRNAs Are Indispensable for Reprogramming Mouse Embryonic Fibroblasts into Induced Stem Cell-Like Cells 
PLoS ONE  2012;7(6):e39239.
MicroRNAs play a pivotal role in cellular maintenance, proliferation, and differentiation. They have also been implicated to play a key role in disease pathogenesis, and more recently, cellular reprogramming. Certain microRNA clusters can enhance or even directly induce reprogramming, while repressing key proteins involved in microRNA processing decreases reprogramming efficiency. Although microRNAs clearly play important roles in cellular reprogramming, it remains unknown whether microRNAs are absolutely necessary. We endeavored to answer this fundamental question by attempting to reprogram Dicer-null mouse embryonic fibroblasts (MEFs) that lack almost all functional microRNAs using a defined set of transcription factors. Transduction of reprogramming factors using either lentiviral or piggyBac transposon vector into two, independently derived lines of Dicer-null MEFs failed to produce cells resembling embryonic stem cells (ESCs). However, expression of human Dicer in the Dicer-null MEFs restored their reprogramming potential. Our study demonstrates for the first time that microRNAs are indispensable for dedifferentiation reprogramming.
doi:10.1371/journal.pone.0039239
PMCID: PMC3380844  PMID: 22737231
6.  Meta-analysis of human lung cancer microRNA expression profiling studies comparing cancer tissues with normal tissues 
Background
Lung cancer is the major cause of cancer death globally, it is often diagnosed at an advanced stage and has one of the lowest survival rates of any type of cancer. The common interest in the field of lung cancer research is the identification of biomarkers for early diagnosis and accurate prognosis. There is increasing evidence to suggest that microRNAs play important and complex roles in lung cancer.
Methods
A meta-analysis was conducted to review the published microRNA expression profiling studies that compared the microRNAs expression profiles in lung cancer tissues with those in normal lung tissues. A vote-counting strategy that considers the total number of studies reporting its differential expression, the total number of tissue samples used in the studies and the average fold change was employed.
Results
A total of 184 differentially expressed microRNAs were reported in the fourteen microRNA expression profiling studies that compared lung cancer tissues with normal tissues, with 61 microRNAs were reported in at least two studies. In the panel of consistently reported up-regulated microRNAs, miR-210 was reported in nine studies and miR-21 was reported in seven studies. In the consistently reported down-regulated microRNAs, miR-126 was reported in ten studies and miR-30a was reported in eight studies. Four up-regulated microRNAs (miR-210, miR-21, miR-31 and miR-182) and two down-regulated mcroiRNAs (miR-126 and miR-145) were consistently reported both in squamous carcinoma and adenocarcinoma-based subgroup analysis, with the other 14 microRNAs solely reported in one or the other subset.
Conclusions
In conclusion, the top two most consistently reported up-regulated microRNAs were miR-210 and miR-21. The results of this meta-analysis of human lung cancer microRNA expression profiling studies might provide some clues of the potential biomarkers in lung cancer. Further mechanistic and external validation studies are needed for their clinical significance and role in the development of lung cancer.
doi:10.1186/1756-9966-31-54
PMCID: PMC3502083  PMID: 22672859
MicroRNAs; Profiling; Lung cancer; Meta-analysis
7.  The Akt-associated microRNAs 
As master gene regulators, microRNAs impact diverse cellular pathways. It is well known that microRNAs are often dysregulated in many types of cancer and other human diseases. In cancer, microRNAs may function as oncogenes or tumor suppressors. Of considerable interest, recent evidence suggests that microRNA-mediated gene regulation interconnects with the Akt pathway, forming an Akt-microRNA regulatory network. MicroRNAs and Akt in this network work together to exert their cellular functions. Thus, a better understanding of this Akt-microRNA regulatory network is critical to successful targeting of the PI3K/Akt pathway for cancer therapy. In this review, we will update on recent advances of how microRNAs impact the Akt activity as well as how microRNAs are regulated through the Akt pathway. We will also briefly discuss the clinical implication of gene regulation mediated through the Akt-associated microRNAs.
doi:10.1007/s00018-012-1129-8
PMCID: PMC3475760  PMID: 22936352
8.  miR-449a Regulates the Chondrogenesis of Human Mesenchymal Stem Cells Through Direct Targeting of Lymphoid Enhancer-Binding Factor-1 
Stem Cells and Development  2012;21(18):3298-3308.
microRNAs are small molecules, about 17–23 nucleotides in length, that act as translational regulators of their target gene. By binding to a target, microRNAs are known to either inhibit translation or induce degradation of the target. Despite the great interest in microRNAs, however, the exact targets of each individual microRNA in different processes remain largely unknown. In this study, we determined that the lymphoid enhancer-binding factor-1 (LEF-1) was expressed during the chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and sought to identify a novel microRNA targeting this gene. Through subsequent studies, we have identified, for the first time, one particular microRNA, miR-449a, that recognizes and regulates the expression of LEF-1 in a dose-dependent and sequence-specific manner. In addition, we observed that the inhibition of LEF-1 via miR-449a led to the subsequent repression of Sox 9, which is a well-established regulator of chondrogenesis. Collectively, this study demonstrated that miR-449a directly targets LEF-1, which in turn affects the expression of Sox 9, ultimately leading to the proper regulation of the differentiation and chondrogenesis of human MSCs (hBM-MSCs).
doi:10.1089/scd.2011.0732
PMCID: PMC3516419  PMID: 22769578
9.  Nuclear Hormone Receptor Regulation of microRNAs Controls Developmental Progression 
Science (New York, N.Y.)  2009;324(5923):95-98.
In response to small molecule signals such as retinoids or steroids, nuclear receptors activate gene expression to regulate development in different tissues. microRNAs turn off target gene expression within cells by binding complementary regions in mRNA transcripts, and they have been broadly implicated in development and disease. Here we show that the C. elegans nuclear receptor DAF-12 and its steroidal ligand directly activate promoters of let-7 microRNA family members to downregulate the microRNA target hbl-1 and drive progression of epidermal stem cells from second to third larval stage patterns of cell division. Conversely, the unliganded receptor represses microRNA expression during developmental arrest. These findings identify microRNAs as components of a hormone-coupled molecular switch that shuts off earlier developmental programs to allow for later ones.
doi:10.1126/science.1164899
PMCID: PMC2757405  PMID: 19342589
10.  Effect of (S)-3,5-DHPG on MicroRNA Expression in Mouse Brain 
Experimental Neurology  2012;235(2):497-507.
MicroRNAs are small non-coding RNAs that regulate post-transcriptional gene expression. In the short time since the discovery of microRNAs, the literature has burgeoned with studies focused on the biosynthesis of microRNAs, target prediction and binding, and mechanisms of translational repression by microRNAs. Given the prominent role of microRNAs in all areas of cell biology, it is not surprising that microRNAs are also linked to human diseases, including those of the nervous system. One of the least-studied areas of microRNA research is how their expression is regulated outside of development and cancer. Thus, we examined a role for regulation of microRNAs by neurotransmitter receptor activation in mouse brain. We focused on the group I metabotropic glutamate receptors by using intracerebroventricular injection of the selective agonist, (S)-3,5-dihydroxyphenylglycine (DHPG) in mouse brain. We then examined the expression of microRNAs in the cerebral cortex by Ambion and Invitrogen microarrays, and the expression of mature microRNA sequences by SABiosciences qPCR arrays, at 4, 8 and 24 hours after DHPG injection. These studies revealed that the largest number of significantly regulated microRNAs was detected 8 hours after DHPG injection in the microarrays and qPCR arrays. We then used RNA blots to quantify microRNA expression, and in situ hybridization to examine cellular distribution of the microRNAs regulated by DHPG. Bioinformatic analysis of the microRNAs regulated 8 hours after DHPG in all three arrays revealed KEGG pathways that are known to correlate with group I mGluR effects, as well as recently described and novel pathways. These studies are the first to show that DHGP regulates the expression of microRNAs in mouse cerebral cortex, and support the hypothesis that group I mGluRs may regulate microRNA expression in mouse brain.
doi:10.1016/j.expneurol.2012.01.018
PMCID: PMC3345043  PMID: 22309833
11.  Direct Conversion of Fibroblasts to Neurons by Reprogramming PTB-Regulated microRNA Circuits 
Cell  2013;152(1-2):82-96.
Summary
The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell lineage-specific transcription factors. Here we report that repression of a single RNA binding protein PTB, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby de-repressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in non-neuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage.
doi:10.1016/j.cell.2012.11.045
PMCID: PMC3552026  PMID: 23313552
12.  Transdifferentiation-inducing HCCR-1 oncogene 
BMC Cell Biology  2010;11:49.
Background
Cell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. The differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules.
Results
We report here that HCCR-1, previously shown to play an oncogenic role in human cancers, induces epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) in human and mouse, respectively. The stem cell factor receptor CD117/c-Kit was induced in this transdifferentiated (EMT) sarcoma tissues. This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis. Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney. This suggests that HCCR-1 might be involved in the transdifferentiation process of cancer stem cell.
Conclusions
Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.
doi:10.1186/1471-2121-11-49
PMCID: PMC2909153  PMID: 20591135
13.  Looking into the Black Box: Insights into the Mechanisms of Somatic Cell Reprogramming 
Genes  2011;2(1):81-106.
The dramatic discovery that somatic cells could be reprogrammed to induced pluripotent stem cells (iPSCs), by the expression of just four factors, has opened new opportunities for regenerative medicine and novel ways of modeling human diseases. Extensive research over the short time since the first iPSCs were generated has yielded the ability to reprogram various cell types using a diverse range of methods. However the duration, efficiency, and safety of induced reprogramming have remained a persistent limitation to achieving a robust experimental and therapeutic system. The field has worked to resolve these issues through technological advances using non-integrative approaches, factor replacement or complementation with microRNA, shRNA and drugs. Despite these advances, the molecular mechanisms underlying the reprogramming process remain poorly understood. Recently, through the use of inducible secondary reprogramming systems, researchers have now accessed more rigorous mechanistic experiments to decipher this complex process. In this review we will discuss some of the major recent findings in reprogramming, pertaining to proliferation and cellular senescence, epigenetic and chromatin remodeling, and other complex cellular processes such as morphological changes and mesenchymal-to-epithelial transition. We will focus on the implications of this work in the construction of a mechanistic understanding of reprogramming and discuss unexplored areas in this rapidly expanding field.
doi:10.3390/genes2010081
PMCID: PMC3924844
induced pluripotent stem cells; mechanism of reprogramming; iPS; BMP; Wnt; p53; epigenetic; MET; PKA; histone modifications
14.  microRNA regulation of neural stem cells and neurogenesis 
MicroRNAs are a class of small RNA regulators that are involved in numerous cellular processes, including development, proliferation, differentiation, and plasticity. The emerging concept is that microRNAs play a central role in controlling the balance between stem cell self-renewal and fate-determination by regulating the expression of stem cell regulators. This review will highlight recent advances in the regulation of neural stem cell self-renewal and neurogenesis by microRNAs. It will cover microRNA functions during the entire process of neurogenesis, from neural stem cell self-renewal and fate-determination to neuronal maturation, synaptic formation, and plasticity. The interplay between microRNAs and both cell-intrinsic and extrinsic stem cell players, including transcription factors, epigenetic regulators, and extrinsic signaling molecules will be discussed. This is a summary of the topics covered in the mini-symposium on microRNA regulation of neural stem cells and neurogenesis in SFN 2010 and is not meant to be a comprehensive review of the subject.
doi:10.1523/JNEUROSCI.4280-10.2010
PMCID: PMC3071711  PMID: 21068294
15.  A New Role for the GARP Complex in MicroRNA-Mediated Gene Regulation 
PLoS Genetics  2013;9(11):e1003961.
Many core components of the microRNA pathway have been elucidated and knowledge of their mechanisms of action actively progresses. In contrast, factors with modulatory roles on the pathway are just starting to become known and understood. Using a genetic screen in Caenorhabditis elegans, we identify a component of the GARP (Golgi Associated Retrograde Protein) complex, vps-52, as a novel genetic interactor of the microRNA pathway. The loss of vps-52 in distinct sensitized genetic backgrounds induces the enhancement of defective microRNA-mediated gene silencing. It synergizes with the core microRNA components, alg-1 Argonaute and ain-1 (GW182), in enhancing seam cell defects and exacerbates the gene silencing defects of the let-7 family and lsy-6 microRNAs in the regulation of seam cell, vulva and ASEL neuron development. Underpinning the observed genetic interactions, we found that VPS-52 impinges on the abundance of the GW182 proteins as well as the levels of microRNAs including the let-7 family. Altogether, we demonstrate that GARP complex fulfills a positive modulatory role on microRNA function and postulate that acting through GARP, vps-52 participates in a membrane-related process of the microRNA pathway.
Author Summary
The microRNA pathway is a post-transcriptional gene regulatory system that uses small non-coding RNAs called microRNAs to control multiple developmental and physiological processes. With the goal of unveiling factors modulating this regulatory pathway, we have undertaken the exploration of genetic interactors in the roundworm Caenorhabditis elegans. We identify vps-52, a component of the Golgi Associated Retrograde Protein or GARP complex, and establish that this complex executes a positive modulatory role on microRNA activity. The absence of vps-52 function exacerbates diverse microRNA-related defects. Molecularly, this effect relates to decreased abundance of microRNAs and the GW182 proteins. Considering that GARP is involved in the traffic of vesicles from endosomes back to the Golgi apparatus, we propose that GARP facilitates a membrane-related process of the microRNA pathway.
doi:10.1371/journal.pgen.1003961
PMCID: PMC3820791  PMID: 24244204
16.  A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts 
Genome Biology  2012;13(12):R121.
Background
Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.
Results
Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.
Conclusions
microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.
doi:10.1186/gb-2012-13-12-r121
PMCID: PMC3924601  PMID: 23259597
MicroRNA; Quiescence; Cell cycle; Proliferation; Extracellular matrix; Fibroblast; Microarray; miR-29
17.  MicroRNAs in Immune Regulation - Opportunities for Cancer Immunotherapy 
Endogenously produced microRNAs are predicted to regulate the translation of over two-thirds all human gene transcripts. Certain microRNAs regulate expression of genes that are critically involved in both innate and adaptive immune responses. Immune cells represent a highly attractive target for microRNA gene therapy approaches, as these cells can be isolated, treated and then reintroduced into the patient. In this short review, we discuss how recent discoveries on the roles of microRNAs in immune-regulation will advance the field of cancer immunology and immunotherapy. Targets identified already in T cells include microRNAs, miR-17-92 family, miR-155, and miR-181a. In macrophages, miR-125b, miR-146, and miR-155 act as Pathogen Associated Molecular Pattern Molecule-associated microRNAs and miR-34C and miR-214 as Damage Associated Molecular Pattern Molecules-associated miRs. We have also demonstrated that the ability of tumors to serve as targets for cytolytic effectors is regulated by miR-222 and miR-339.
doi:10.1016/j.biocel.2010.02.002
PMCID: PMC2889234  PMID: 20144731
microRNA; Type-1 helper (Th1); cancer; cancer immunology; high-mobility group box (HMGB)1
18.  Similarity-based methods for potential human microRNA-disease association prediction 
BMC Medical Genomics  2013;6:12.
Background
The identification of microRNA-disease associations is critical for understanding the molecular mechanisms of diseases. However, experimental determination of associations between microRNAs and diseases remains challenging. Meanwhile, target diseases need to be revealed for some new microRNAs without any known target disease association information as new microRNAs are discovered each year. Therefore, computational methods for microRNA-disease association prediction have gained a lot of research interest.
Methods
Herein, based on the assumption that functionally related microRNAs tend to be associated with phenotypically similar diseases, three inference methods were presented for microRNA-disease association prediction, namely MBSI (microRNA-based similarity inference), PBSI (phenotype-based similarity inference) and NetCBI (network-consistency-based inference). Global network similarity measure was used in the three methods to predict new microRNA-disease associations.
Results
We tested the three methods on 242 known microRNA-disease associations by leave-one-out cross-validation for prediction evaluation, and achieved AUC values of 74.83%, 54.02% and 80.66%, respectively. The best-performed method NetCBI was then chosen for novel microRNA-disease association prediction. Some associations strongly predicted by NetCBI were confirmed by the publicly accessible databases, which indicated the usefulness of this method. The newly predicted associations were publicly released to facilitate future studies. Moreover, NetCBI was especially applicable to predicting target diseases for microRNAs whose target association information was not available.
Conclusions
The encouraging results suggest that our method NetCBI can not only provide help in identifying novel microRNA-disease associations but also guide biological experiments for scientific research.
doi:10.1186/1755-8794-6-12
PMCID: PMC3629999  PMID: 23570623
MicroRNA-disease association prediction; Network similarity; Network consistency
19.  Blockage of transdifferentiation from fibroblast to myofibroblast in experimental ovarian cancer models 
Molecular Cancer  2009;8:78.
Background
Tumour stromal myofibroblasts can promote tumour invasion. As these cells are genetically more stable than cancer cells, there has been enormous interest in developing targeted molecular therapies against them. Chloride intracellular channel 4 (CLIC4) and reactive oxygen species (ROS) have been linked with promoting stromal cell transdifferentiation in various cancers, but little is known of their roles in ovarian cancer. In this study, we examined the functional roles that both CLIC4 and ROS play in the process of ovarian cancer cell-stimulated or TGF-β1 induced fibroblast-to-myofibroblast transdifferentiation. We also examine whether it is possible to reverse such a process, with the aim of developing novel therapies against ovarian cancer by targeting activated transdifferentiated myofibroblasts.
Results
We demonstrate that TGF-β1 induced or CMSKOV3 activate transdifferentiated myofibroblasts (fibroblasts). These fibroblasts mimic "reactive" stromal myofibroblasts and demonstrate significant up-regulation of CLIC4 expression and increased level of ROS production. Blocking the production of ROS with an antioxidant consequently reduces the expression of CLIC4, and is accompanied by disappearance of α-smooth-muscle actin (α-SMA), a myofibroblast marker, suggesting ROS acts as a signalling molecule that promotes and enhances CLIC4 activities in the myofibroblast transdifferentiaton process. Down-regulation of CLIC4 with a generic agent or specific siRNA both significantly reduces the expression of factors related to the phenotypes and functions of myofibroblasts, such as α-SMA, hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), thus reversing the myofibroblast phenotype back to fibroblasts. These results convincingly show that ROS and CLIC4 are responsible for TGF-β1 induced fibroblast-to-myofibroblast transdifferentiaton and down-regulation of both is sufficient to block transdifferentiated myofibroblasts.
Conclusion
Molecular targeting of ROS and CLIC4 has the potential to develop novel therapies for ovarian cancer.
doi:10.1186/1476-4598-8-78
PMCID: PMC2765417  PMID: 19781102
20.  Neuronal Activity Rapidly Induces Transcription of the CREB-Regulated microRNA-132, in vivo 
Hippocampus  2010;20(4):492-498.
Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its expression in mouse brain was monitored by quantitative RT-PCR (RT-qPCR). Pilocarpine-induced seizures led to a robust, rapid, and transient increase in the primary transcript of miR-132 (pri-miR-132) followed by a subsequent rise in mature microRNA (miR-132). Activation of neurons in the hippocampus, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 minutes and returning to basal levels within two hours of stimulation. Expression levels of primary and mature-miR-132 increased significantly between postnatal days 10 and 24. We conclude that miR-132 is an activity-dependent microRNA in vivo, and may contribute to the long-lasting proteomic changes required for experience-dependent neuronal plasticity.
doi:10.1002/hipo.20646
PMCID: PMC2847008  PMID: 19557767
MicroRNA; CREB; Plasticity; Experience-dependent; Immediate-early; mir-132
21.  The Impact of Hemolysis on Cell-Free microRNA Biomarkers 
Cell-free microRNAs in plasma and serum have become a promising source of biomarkers for various diseases. Despite rapid progress in this field, there remains a lack of consensus regarding optimal quantification methods, reference genes, and quality control of samples. Recent studies have shown that hemolysis occurring during blood collection has substantial impact on the microRNA content in plasma/serum. To date, the impact of hemolysis has only been investigated for a limited number of microRNAs, mainly the red blood cell (RBC)-enriched miRs-16 and -451. In contrast, the effect of hemolysis on other microRNAs – in particular those proposed as biomarkers – has not been addressed. In this study we profiled the microRNA content of hemolyzed and non-hemolyzed plasma as well as RBCs to obtain a profile of microRNAs in the circulation affected or unaffected by hemolysis. Profiling by TaqMan Array Microfluidic Cards was used to compare three pairs of hemolyzed and non-hemolyzed plasma (with varying degrees of hemolysis) and one RBC sample. A total of 136 microRNAs were detectable in at least two of the samples, and of those 15 were at least twofold elevated in all three hemolyzed samples. This number increased to 88 microRNAs for the sample with the highest level of hemolysis, with all of these also detected in the RBC profile. Thus these microRNAs represent a large proportion of detectable microRNAs and those most likely to be affected by hemolysis. Several of the hemolysis-susceptible microRNAs (e.g., miRs-21, -106a, -92a, -17, -16) have also been previously proposed as plasma/serum biomarkers of disease, highlighting the importance of rigorous quality control of plasma/serum samples used for measurement of circulating microRNAs. As low-level hemolysis is a frequent occurrence during plasma/serum collection it is critical that this is taken into account in the measurement of any candidate circulating microRNA.
doi:10.3389/fgene.2013.00094
PMCID: PMC3663194  PMID: 23745127
cell-free microRNA; red blood cells; hemolysis; biomarker; quality control
22.  MicroRNA as a Novel Modulator in Head and Neck Squamous Carcinoma 
Journal of Oncology  2011;2010:135632.
MicroRNAs have emerged as important regulators of cell proliferation, development, cancer formation, stress responses, cell death, and other physiological conditions in the past decade. On the other hand, head and neck cancer is one of the top ten most common cancers worldwide. Recent advances in microRNAs have revealed their prominent role in regulating gene expression and provided new aspects of applications in diagnosis, prognosis, and therapeutic strategies in head and neck squamous carcinoma. In the present paper, we focus on microRNAs showing significant differences between normal and tumor cells or between cells with differential ability of metastasis. We also emphasize specific microRNAs that could modulate tumor cell properties, such as apoptosis, metastasis, and proliferation. These microRNAs possess the potential to be applied on clinical therapy in the future.
doi:10.1155/2010/135632
PMCID: PMC3065009  PMID: 21461395
23.  Microarray analysis of microRNA expression in the developing mammalian brain 
Genome Biology  2004;5(9):R68.
A microarray technology suitable for analyzing the expression of microRNAs and of other small RNAs was used to determine the microRNA expression profile during mouse-brain development and observed a temporal wave of gene expression of sequential classes of microRNAs.
Background
MicroRNAs are a large new class of tiny regulatory RNAs found in nematodes, plants, insects and mammals. MicroRNAs are thought to act as post-transcriptional modulators of gene expression. In invertebrates microRNAs have been implicated as regulators of developmental timing, neuronal differentiation, cell proliferation, programmed cell death and fat metabolism. Little is known about the roles of microRNAs in mammals.
Results
We isolated 18-26 nucleotide RNAs from developing rat and monkey brains. From the sequences of these RNAs and the sequences of the rat and human genomes we determined which of these small RNAs are likely to have derived from stem-loop precursors typical of microRNAs. Next, we developed a microarray technology suitable for detecting microRNAs and printed a microRNA microarray representing 138 mammalian microRNAs corresponding to the sequences of the microRNAs we cloned as well as to other known microRNAs. We used this microarray to determine the profile of microRNAs expressed in the developing mouse brain. We observed a temporal wave of expression of microRNAs, suggesting that microRNAs play important roles in the development of the mammalian brain.
Conclusion
We describe a microarray technology that can be used to analyze the expression of microRNAs and of other small RNAs. MicroRNA microarrays offer a new tool that should facilitate studies of the biological roles of microRNAs. We used this method to determine the microRNA expression profile during mouse brain development and observed a temporal wave of gene expression of sequential classes of microRNAs.
doi:10.1186/gb-2004-5-9-r68
PMCID: PMC522875  PMID: 15345052
24.  MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells 
Nature methods  2007;4(9):10.1038/nmeth1079.
MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed ‘microRNA sponges’, these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.
doi:10.1038/nmeth1079
PMCID: PMC3857099  PMID: 17694064
25.  MicroRNAs and lung cancer: Biology and applications in diagnosis and prognosis 
MicroRNAs are tiny non-coding RNA molecules which play important roles in the epigenetic control of cellular processes by preventing the translation of proteins from messenger RNAs (mRNAs). A single microRNA can target different mRNAs, and an mRNA can be targeted by multiple microRNAs. Such complex interplays underlie many molecular pathways in cells, and specific roles for many microRNAs in physiological as well as pathological phenomena have been identified. Changes in expression of microRNAs have been associated with a wide variety of disease conditions, and microRNA-based biomarkers are being developed for the identification and monitoring of such states. This review provides a general overview of the current state of knowledge about the biology of microRNAs, and specific information about microRNAs with regard to the diagnosis and prognosis of lung cancer.
doi:10.4103/1477-3163.67074
PMCID: PMC2925280  PMID: 20808843
Carcinogenesis; gene expression; microRNA; lung cancer

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