Putative miRNA Research Articles

Comparative Profiling of miRNAs and Target Gene Identification in Distant-Grafting between Tomato and Lycium (Goji Berry).

Local translocation of small RNAs between cells is proved. Long distance translocation between rootstock and scion is also well documented in the homo-grafting system, but the process in distant-grafting is widely unexplored where rootstock and scion belonging to different genera. Micro RNAs are a class of small, endogenous, noncoding, gene silencing RNAs that regulate target genes of a wide range of important biological pathways in plants. In this study, tomato was grafted onto goji (Lycium chinense Mill.) to reveal the insight of miRNAs regulation and expression patterns within a distant-grafting system. Goji is an important traditional Chinese medicinal plant with enriched phytochemicals. Illumina sequencing technology has identified 68 evolutionary known miRNAs of 37 miRNA families. Moreover, 168 putative novel miRNAs were also identified. Compared with control tomato, 43 (11 known and 32 novels) and 163 (33 known and 130 novels) miRNAs were expressed significantly different in shoot and fruit of grafted tomato, respectively. The fruiting stage was identified as the most responsive in the distant-grafting approach and 123 miRNAs were found as up-regulating in the grafted fruit which is remarkably higher compare to the grafted shoot tip (28). Potential targets of differentially expressed miRNAs were found to be involved in diverse metabolic and regulatory pathways. ADP binding activities, molybdopterin synthase complex and RNA helicase activity were found as enriched terms in GO (Gene Ontology) analysis. Additionally, “metabolic pathways” was revealed as the most significant pathway in KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. The information of the small RNA transcriptomes that are obtained from this study might be the first miRNAs elucidation for a distant-grafting system, particularly between goji and tomato. The results from this study will provide the insights into the molecular aspects of miRNA-mediated regulation in the medicinal plant goji, and in grafted tomato. Noteworthy, it would provide a basis how miRNA signals could exchange between rootstock and scion, and the relevance to diverse biological processes.

Novel microRNA discovery using small RNA sequencing in post-mortem human brain.

BackgroundMicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression mainly through translational repression of target mRNA molecules. More than 2700 human miRNAs have been identified and some are known to be associated with disease phenotypes and to display tissue-specific patterns of expression.MethodsWe used high-throughput small RNA sequencing to discover novel miRNAs in 93 human post-mortem prefrontal cortex samples from individuals with Huntington’s disease (n = 28) or Parkinson’s disease (n = 29) and controls without neurological impairment (n = 36). A custom miRNA identification analysis pipeline was built, which utilizes miRDeep* miRNA identification and result filtering based on false positive rate estimates.ResultsNinety-nine novel miRNA candidates with a false positive rate of less than 5 % were identified. Thirty-four of the candidate miRNAs show sequence similarity with known mature miRNA sequences and may be novel members of known miRNA families, while the remaining 65 may constitute previously undiscovered families of miRNAs. Nineteen of the 99 candidate miRNAs were replicated using independent, publicly-available human brain RNA-sequencing samples, and seven were experimentally validated using qPCR.ConclusionsWe have used small RNA sequencing to identify 99 putative novel miRNAs that are present in human brain samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3114-3) contains supplementary material, which is available to authorized users.

Analysis of high iron rice lines reveals new miRNAs that target iron transporters in roots.

The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants . Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds.

Immediate-early alcohol-responsive miRNA expression in Drosophila

At the core of the changes characteristic of alcoholism are alterations in gene expression in the brain of the addicted individual. These changes are believed to underlie some of the neuroadaptations that promote compulsive drinking. Unfortunately, the mechanisms by which alcohol consumption produces changes in gene expression remain poorly understood. MicroRNAs (miRNAs) have emerged as important regulators of gene expression because they can coordinately modulate the translation efficiency of large sets of specific mRNAs. Here, we investigate the early miRNA responses elicited by an acute sedating dose of alcohol in the Drosophila model organism. In our analysis, we combine the power of next-generation sequencing with Drosophila genetics to identify alcohol-sensitive miRNAs and to functionally test them for a role in modulating alcohol sensitivity. We identified 14 known Drosophila miRNAs, and 13 putative novel miRNAs that respond to an acute sedative exposure to alcohol. Using the GeneSwitch Gal4/UAS system, a subset of these ethanol-responsive miRNAs was functionally tested to determine their individual contribution in modulating ethanol sensitivity. We identified two microRNAs that when overexpressed significantly increased ethanol sensitivity: miR-6 and miR-310. MicroRNA target prediction analysis revealed that the different alcohol-responsive miRNAs target-overlapping sets of mRNAs. Alcoholism is the product of accumulated cellular changes produced by chronic ethanol consumption. Although all of the changes described herein are extremely rapid responses evoked by a single ethanol exposure, understanding the gene expression changes that occur in the first few minutes after ethanol exposure will help us to categorize ethanol responses into those that are near instantaneous and those that are emergent responses produced only by repeated ethanol exposure.

Integrated Data Set of microRNAs and mRNAs Involved in Severe Intrauterine Adhesion.

Adhesion tissue is formed following injury to the uterine basal layer. Currently, there is no effective treatment for severe intrauterine adhesion (IUA), which causes loss of reproductive function. Enhanced understanding of the molecular mechanisms driving severe IUA would be beneficial for the treatment. Differentially expressed microRNAs (miRNAs) and messenger RNAs (mRNAs) in severe IUA (n = 3) and normal (n = 3) endometrium were analyzed by high-throughput microarray analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and found to overlap with the differentially expressed mRNAs. Gene Ontology and pathway analyses were performed for the intersecting genes. Three of the significantly dysregulated miRNAs and 4 of their target mRNAs were further assessed using quantitative real-time polymerase chain reaction (PCR) in 10 severe IUA and 10 normal endometrium samples. Microarray analysis indicated that 26 miRNAs and 1180 mRNAs were significantly different between the 2 groups. Of these, 16 miRNAs and 54 mRNAs overlapped with putative miRNA target genes and prediction of target gene. Real-time PCR revealed upregulation of hsa-miR-513a-5p and has-miR-135a-3p and downregulation of hsa-miR-543 and their corresponding target genes, plus downregulation of ADAM9 (a disintegrin-containing and metalloproteinases) and lysyl oxidase and upregulation of CDH2 (N-cadherin) and COL16A1 (collagen 16A1). Both CDH2 and COL16A1 were bioinformatically predicted and confirmed in vitro as target genes of miR-543. This study provides an integrated data set of the miRNA and mRNA profiles in severe IUA, showing involvement of many miRNAs and their target genes. Further analysis of these genes will help in understanding of the molecular mechanism of IUA formation.

MiR-155–regulated molecular network orchestrates cell fate in the innate and adaptive immune response to Mycobacterium tuberculosis

The regulation of host-pathogen interactions during Mycobacterium tuberculosis (Mtb) infection remains unresolved. MicroRNAs (miRNAs) are important regulators of the immune system, and so we used a systems biology approach to construct an miRNA regulatory network activated in macrophages during Mtb infection. Our network comprises 77 putative miRNAs that are associated with temporal gene expression signatures in macrophages early after Mtb infection. In this study, we demonstrate a dual role for one of these regulators, miR-155. On the one hand, miR-155 maintains the survival of Mtb-infected macrophages, thereby providing a niche favoring bacterial replication; on the other hand, miR-155 promotes the survival and function of Mtb-specific T cells, enabling an effective adaptive immune response. MiR-155-induced cell survival is mediated through the SH2 domain-containing inositol 5-phosphatase 1 (SHIP1)/protein kinase B (Akt) pathway. Thus, dual regulation of the same cell survival pathway in innate and adaptive immune cells leads to vastly different outcomes with respect to bacterial containment.

Variations in the Genome: The Mutation Detection 2015 Meeting on Detection, Genome Sequencing, and Interpretation.

The content of the 13th Mutation Detection meeting (Leiden, April 2015) is summarized in this report. Topics discussed at the meeting included current challenges of clinical NGS, advances in bioinformatics, data quality control, single cell analysis and RNA sequencing, among others. Social, ethical and regulatory challenges of genomic data handling and data sharing were the focus of an expert panel debate. The 14th International Symposium on Variants in the Genome will take place in Santiago de Compostela, June 5-8, 2017. http://isv.variome.org.

Identification and characterization of miRNAs involved in adventitious branches formation of Gracilaria lichenoides in vitro

The main morphological response of Gracilaria lichenoides explants to wounding in vitro was the direct production of adventitious branches (ABs) within a short period rather than callus formation, similar to other seaweeds. However, the underlying mechanism remains unclear because of the lack of genome information. MicroRNAs (miRNAs) play a role in plant growth and development by targeting mRNAs for translational repression or cleavage. Here, we investigated small RNA-mediated regulation of ABs formation by examining the post-transcriptional profiles of different treatment samples of G. lichenoides using the Illumina sequencing platform. The results showed that miR156g-3p, miR2597, miR3447-3p, miR169j-3p and other miRNAs families play significant roles in ABs formation in G. lichenoides through their predicted targets, including phospholipase D (PLD), auxin response factor (ARF), protein phosphatase 2C (PP2C), CTR1 and adenylate isopentenyltransferase (IPT). In addition, we identified 30 putative unique Gracilariales miRNAs in two small RNA libraries that may be involved in ABs induction. Profiling of miRNAs and their target genes of G. lichenoides provide new insight on miRNAs networks involved in ABs formation in seaweed.

Identification of conserved and novel microRNAs in Porphyridium purpureum via deep sequencing and bioinformatics.

BackgroundPorphyridium purpureum has been utilized in important industrial and pharmaceutical fields. The identification of microRNAs (miRNAs) in this unique species is of great importance: such identification can help fill gaps in the small RNA (sRNA) studies of this organism and help to elucidate essential biological processes and their regulation mechanisms in this special micro alga.ResultsIn this study, 254 high-confidence miRNAs (203 conserved miRNAs and 51 novel miRNAs) were identified by sRNA deep sequencing (sRNA-seq) combined with bioinformatics. A total of 235 putative miRNA families were predicted, including 192 conserved families and 43 species-specific families. The conservation and diversity of predicted miRNA families were analysed in different plant species. Both the 100 % northern blot validation rate (VR) of four randomly selected miRNAs and the results of stem-loop quantitative real time RT-PCR (qRT-PCR) assays of 25 randomly selected miRNAs demonstrated that the majority of the miRNAs identified in this study are credible. A total of 14,958 and 2184 genes were predicted to be targeted by the 186 conserved and 41 novel miRNAs. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that some target genes likely provide valuable references for further understanding of vital functions in P. purpureum. In addition, a cytoscape network will provide some clues for research into the complex biological processes that occur in this unique alga.ConclusionsWe first identified a large set of conserved and novel miRNAs in P. purpureum. The characteristic and validation analysis on miRNAs demonstrated authenticity of identification data. Functional annotation of target genes and metabolic pathways they involved in illuminated the direction for further utilization and development this micro alga based on its unique properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2985-7) contains supplementary material, which is available to authorized users.

Identification of potential miRNAs and candidate genes of cervical intraepithelial neoplasia by bioinformatic analysis.

The objective of this study was to predict potential target genes and key miRNAs for cervical intraepithelial neoplasia (CIN) by bioinformatics analysis. The microarray data of GSE51993 were downloaded from Gene Expression Omnibus (GEO) database. Total 30 chips data from two platforms (each platform including eight CIN III samples data and seven normal cervix samples data) were used to identify the feature miRNAs and genes between CIN III and normal samples, respectively. Then the miRNA- mRNA regulatory network was constructed using Cytoscape software. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for all target genes with the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool. Transcription factors (TFs) and cancer-related genes were analyzed. Total 21 putative target miRNAs and 361 putative target mRNAs were gained. The miRNA-mRNA regulatory network results showed that miR-338-5p, miR-193a-5p, and miR-216b were top three hub nodes. GO terms significantly enriched were extracellular region (p = 0.004191) and embryonic skeletal system (p = 0.004742). No significantly enriched KEGG pathway term was found in this study. PBXl (pre-B-cell leukemia transcription factor 1) and LAMC2 (laminin subunit gamma-2) were cancer-promoting genes and also, PBX was TF. PBXl and LAMC2 may be target genes for CIN. MiR-338, and miR-216 may be key miRNAs in CIN development.

Identification of Known and Novel microRNAs and Their Targets in Peach (Prunus persica) Fruit by High-Throughput Sequencing.

MicroRNAs (miRNAs) are a group of non-coding RNAs that have functions in post-transcriptional gene regulation in plants. Although the most important economic component of peach trees (Prunus persica) is the fruit, not much is known about miRNAs in this organ. In this study, miRNAs and their targets were identified and characterized from libraries of small RNAs of peach fruit through Solexa based-sequencing and bioinformatics approaches. A total of 557 known peach miRNAs belonging to 34 miRNA families were identified, and some of these miRNAs were found to be highly conserved in at least four other plant species. Using the most current criteria for miRNA annotation, 275 putative novel miRNAs were predicted, and the sequencing frequencies of these novel miRNAs were less than those of the conserved miRNAs. In total, 3959 and 1614 target genes for 349 known and 193 novel miRNAs, respectively, were predicted with the criteria that a single target gene can be targeted by different miRNAs and that a single miRNA can also have a large number of target genes. Three targets were even found to be targeted by 13 novel miRNAs that contained the same complete miRNA sequence at different locations and had different scaffolds. The proteins predicted to be targeted by the miRNAs identified in this study encompass a wide range of transcription factors and are involved in many biological processes and pathways, including development, metabolism, stress responses and signal transduction. A total of 115 and 101 target genes were identified to be cleaved by 60 known miRNAs and 27 novel miRNAs through degradome sequencing, respectively. These miRNAs induce cleavage of their targets precisely at the position between nucleotides 10 and 11 of the miRNA sequences from the 5’ to the 3’ end. Thirty conserved miRNAs and 19 novel miRNAs exhibited differential expression profiles in the peach, and the expression patterns of some miRNAs appeared to be tissue- or developmental stage-specific. The findings of this study provide an important basis for the analysis of miRNAs, their targets and the functions of these targets in peach fruit.

Abstract 1120: Differential expression of human cytomegalovirus microRNA in triple-negative breast cancer tumors

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of the breast cancer cases. These tumors are heterogeneous and aggressive, fail to respond to targeted therapy, have poor prognosis, and a high risk of relapse. The molecular basis of this breast cancer subtype is currently unknown. Viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human papillomavirus (HPV), are major suspects in the etiology of breast cancer. Since miRNAs have been implicated in the pathogenesis of breast cancer, including triple-negative breast cancer, we hypothesized that viral miRNAs may play a role in this aggressive disease. The objective of this project, therefore, was to determine the identity, prevalence, sequence variation, and differential expression of viral miRNAs in TNBC tumors as compared to control samples. We conducted a comprehensive profiling of viral miRNAs in 48 TNBC tumors as compared to 15 control normal breast tissues, utilizing deep sequencing analysis software and publically available deep sequencing data. Five novel putative HCMV miRNAs were found to be differentially expressed in TNBC tumors as compared to controls. Two of the putative miRNAs were differentially expressed in 60-79% of the TNBC tumors as compared to 0% of normal controls. Two additional putative miRNAs were expressed in 67-88% of TNBC tumors as compared to 25-31% of normal controls. One putative miRNA was expressed in 67% of TNBC tumors as compared to 88% of normal samples, while 1 putative miRNA in addition to HCMV-US25-1-3p displayed no significant differences. These putative HCMV miRNAs localize within genomic regions of HCMV that encode UL56 DNA packaging terminase subunit 2, single-stranded DNA-binding protein, noncoding RNA4.9, and enveloped glycoprotein M. Ongoing work has so far computationally validated one of these miRNAs as a novel HCMV miRNA. This is the first report on the differential expression of HCMV miRNAs in TNBC tumors. Since TNBC tumors are extremely heterogeneous, it is intriguing that 60-80% of TNBC tumors specifically express the same HCMV miRNA. Our findings suggest that these differentially expressed HCMV miRNAs may potentially play a role in the pathogenesis of TNBC. Citation Format: Tia Hudson, Scott Harrison, Dukka K C, Jan Prins, Perpetua M. Muganda. Differential expression of human cytomegalovirus microRNA in triple-negative breast cancer tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1120.

Abstract 1924: miR-142-3p is a candidate mediator of chloride intracellular channel 4 (CLIC4) loss during squamous cancer progression

Abstract Chloride intracellular channel 4 (CLIC4) is a broadly expressed protein that has been implicated in multiple cellular processes, including cellular differentiation and apoptosis. As a soluble factor, CLIC4 translocates to the nucleus following exposure to a variety of stimuli, including TGF-β, TNF-α, and etoposide. Nuclear CLIC4 potentiates TGF-β signaling by preventing the dephosphorylation of phospho-SMAD2 and 3 and promotes growth arrest. Although its primary function is as a tumor suppressor, CLIC4 expression is progressively diminished during cancer progression and CLIC4 protein is excluded from the nucleus of many human epithelial neoplasms and squamous cell carcinoma (SCC) cell lines. To date, no gene deletions or mutations have been identified to account for this phenomenon; thus, identifying the mechanism of CLIC4 loss is our primary focus. To determine if epigenetic gene silencing is responsible, we evaluated the methylation status of the CLIC4 promoter in a progression panel (normal, papilloma, SCC) of epithelial cell lines by performing bisulfite sequencing. No differential promoter methylation was found between the cell lines, suggesting that the regulation may be post-transcriptional or post-translational. Because microRNAs (miRNAs) are known to regulate gene expression by mediating the degradation or translational repression of target mRNAs, we performed a bioinformatic analysis of the CLIC4 3’ UTR, which revealed a large number of putative targeting miRNAs. One such miRNA, miR-142-3p, has been predicted to target CLIC4 by multiple algorithms and has been shown to interact with the CLIC4 3’ UTR by high-throughput sequencing and crosslinking immunoprecipitation. We confirmed the functional activity of miR-142-3p against the CLIC4 3’ UTR by using a 3’ UTR luciferase reporter assay and a miR-142-3p mimic. miR-142-3p is high in the tumor tissue and plasma of patients with head and neck SCC and is correlated with worse prognosis, where CLIC4 expression is known to be low. Thus, miR-142-3p may be responsible for mediating CLIC4 loss in squamous lesions and warrants further investigation. Citation Format: Brandi L. Carofino, Stuart H. Yuspa. miR-142-3p is a candidate mediator of chloride intracellular channel 4 (CLIC4) loss during squamous cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1924.

Abstract 1913: Inhibition of S-Adenosylmethionine-dependent methyltransferase resists TGF-β1-induced EMT of pancreatic cancer via microRNA alterations

Abstract Identification of epigenetic reversal agents for use in combination chemotherapies for human pancreatic ductal adenocarcinomas remains an unmet clinical need. Pharmacological inhibitors of EZH2 are emerging as potential epigenetic reversal agents in various solid tumors and leukemia, however, the surprisingly small set of mRNA transcripts identified with EZH2 knockdown suggests novel mechanisms to govern their anti-tumorigenic effects. Here, we report 3-deazaneplanocin-A, an inhibitor of S-adenosyl-L-homocysteine hydrolase and EZH2 histone lysine-N-methyltransferase, to significantly reprogram the noncoding miRNA expression, including a subset of PDAC-downregulated miRNAs, to dampen TGF-β-induced EMT signals in pancreatic cancer. Particularly, we identify epigenetic silencing of miR-663a and -4787-5p in PDAC tissues and cell lines that are reactivated by 3-deazaneplanocin-A to directly target TGF-β1 for RNA interference. Lentiviral overexpression of miR-663a and -4787-5p partially phenocopied 3-deazaneplanocin-A's EMT resisting effects in pancreatic cancer cells, whereas locked nucleic acid antimiR-663a and -4787-5p mitigated them. In vivo, 3-deazaneplanocin-A, miR-663a and -4787-5p significantly reduced tumor burden and metastasis in an orthotopic mouse pancreatic tumor model with miR-663a displaying more potency than miR-4787-5p in curtailing TGF-β1 signaling pathway. These findings reveal tumor miRNA reprogramming as a novel approach for controlling TGF-β-induced EMT in human PDAC and uncover a list of putative miRNA targets that may guide further selection and development of synthetic histone methylation reversal agents for clinical use. Citation Format: Rajgopal Govindarajan, Hardik Modi. Inhibition of S-Adenosylmethionine-dependent methyltransferase resists TGF-β1-induced EMT of pancreatic cancer via microRNA alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1913.

Carrot plastid terminal oxidase gene (DcPTOX) responds early to chilling and harbors intronic pre-miRNAs related to plant disease defense

The nuclear-encoded plastid terminal oxidase gene (PTOX) is present in photosynthetic species and functions in the oxidation of the plastoquinone pool. It plays an important role on chlororespiration, chromorespiration and carotenoid biosynthesis. Here, we show short-term early response of carrot PTOX (DcPTOX) in leaves of two carrot inbred lines upon chilling. Analysis of the complete gene confirmed DcPTOX as a single gene and revealed an exceptionally large genomic sequence (9422bp) in comparison to other species, comprising nine exons interrupted by eight introns. In silico analysis based on data from whole genome sequencing projects discovered that some plant species present two PTOX genes. A search for sequence variability at genomic level was performed in the heterogeneous, ancient carrot cultivar ‘Rotin’. DcPTOX revealed intron length polymorphisms (ILPs) in intron 2, due to the occurrence of two insertion/deletions (InDels) events. Prediction of pre-miRNA sequences in intronic regions of DcPTOX showed two putative locations coding for putative miRNAs, one located at intron 2 and other located at intron 6. Both putative miRNAs revealed high homology with miRNAs described in Glycine max (gma-miR1520p) and Oryza sativa (osa-miR5494), respectively. Plant disease resistance genes were identified as miRNAs target genes.

Genome-wide identification of microRNAs and their target genes in Cynoglossus semilaevis using computational approach

MicroRNAs (miRNAs) are endogenous non-protein-coding RNAs of approximately 22 nucleotides which regulate gene expression at the posttranscriptional level in plants and animals. Until recently, only limited numbers of miRNAs were identified in Cynoglossus semilaevis, which is an important commercial mariculture fish species in China. The recently sequenced genome of C. semilaevis provides an opportunity to identify some novel miRNAs in this species by computational approach. A total of 217 miRNAs belonging to 87 families were identified in C. semilaevis, including 96 known miRNAs and 121 putative novel miRNAs. To verify the predicted miRNAs, a qRT-PCR-based assay was performed, the results showed that randomly selected 6 miRNAs have different expression patterns in the liver, spleen, kidney and muscles tissues of C. semilaevis, and suggest that these miRNAs might be involved in tissue-specific expression. Following this, 20,614 potential target genes were predicted by searching the mRNA database, and these targets are involved in several key pathways. Finally, the analysis on GO enrichment and KEGG pathway showed that the predicted targets genes for these identified miRNAs participate in diverse physiological and metabolic processes. Taken together, the present study expands the collection of miRNAs and provides a foundation for further understanding the role of these miRNAs in the C. semilaevis.

MiR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of head and neck. Although radiotherapy is used for OSCC treatment, the occurrence of radioresistant cancer cells limits its efficiency. MicroRNAs (miRNAs) are non-coding RNAs with lengths of 18–25 base pairs and known to be involved in carcinogenesis. We previously demonstrated that by targeting B lymphoma Mo-MLV insertion region 1 homolog (Bmi1), miR-494-3p functions as a putative tumor suppressor miRNA in OSCC. In this study, we further discovered that miR-494-3p could enhance the radiosensitivity of SAS OSCC cells and induce cellular senescence. The overexpression of miR-494-3p in SAS cells increased the population of senescence-associated β-galactosidase positive cells, the expression of p16INK4a and retinoblastoma 1 (RB1), as well as downregulated Bmi1. The knockdown of Bmi1 by lentiviral-mediated delivery of specific short hairpin RNAs (shRNAs) also enhanced the radiosensitivity of SAS cells and the activation of the senescence pathway. Furthermore, the inverse correlation between Bmi1 and miR-494-3p expression was observed among OSCC tissues. Results suggest that miR-494-3p could increase the radiosensitivity of OSCC cells through the induction of cellular senescence caused by the downregulation of Bmi1.

MicroRNA-27a-3p regulates epithelial to mesenchymal transition via targeting YAP1 in oral squamous cell carcinoma cells.

MicroRNAs (miRNAs) are small non-coding RNAs frequently dysregulated in human malignancies. Here, we profiled isolated cells from freshly resected tumors from oral squamous cell carcinoma (OSCC) patients and OSCC cell lines using a SYBR Green-based qPCR miRNA array to identify the expression change of the miRNAs. Based on the microarray data and clincopathological factor analysis of 50 OSCC patients related to these miRNAs, miR-27a-3p was selected as a putative miRNA which might play important role in OSCC progression. By bioinformatics analysis and dual-luciferase reporter assay, we found that YAP1 (Yes-associated protein-1) was a direct target gene of miR-27a-3p. Intriguingly, increased expression of miR-27a-3p could significantly decrease the expression level of YAP1 as well as several epithelial to mesenchymal transition (EMT)-related molecules in OSCC cell lines, including Twist and Snail. Then, follow-up studies revealed that miR-27a-3p expression was able to downregulate the EMT-related molecules effectively, which might be involved in the regulation of Sox2 via the YAP1-OCT4-Sox2 signaling axis. In summary, this study found that miR-27a-3p could inhibit the YAP1 directly by post-transcriptionally silencing and potentially suppress EMTprocess, suggesting that miR‑27a-3p might play pivotal roles in effectively manipulating the invasion and metastasis in oral squamous cell carcinoma cells through the EMT inhibition.

Inhibition of Ebola Virus by Anti-Ebola miRNAs in silico.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate transcriptional and posttranscriptional gene regulation of the organisms. miRNA provides immune defense when the body is faced with challenges intracellular agents. miRNA molecules trigger gene silencing in eukaryotic cells. More than 3,000 different human miRNAs (hsa-miRs) have been identified thus far. During ontogenesis, viral or intracellular parasitic infections, miRNAs are differentially expressed to protect the host from intracellular invaders. In a viral infection context, miRNAs have been connected with the interplay between host and pathogen, and occupy a major role in pathogenesis. An in silico approach was used to analyze the four major Ebola Virus genome sequences including the recently characterized Ebola virus responsible for West African epidemic that has killed over 10,000 people. All totaled, 2,543 mature human miRNA sequences were retrieved through an miR-database, and the identification of mature miRNAs were aligned with full length sequences of the four major Ebola viruses via computational tools. We identified 32 miRNAs that exhibited significant inhibitory capacity to block more than one EBV strains. miR-607 showed capacity to quell all four major EBVs. Ten putative miRNAs were found to have near perfect identity at seed sequences with numerous targets of Ebola virus that may completely degrade the viral transcripts. We hypothesize that a miRNA-based vaccine can quell Ebola virus infection. Future approaches will focus on validation of these miRNAs in quelling the Ebola virus to further elucidate their biological functions in primate and other animal models.

SNPs in NRXN1 and CHRNA5 are Associated to Smoking and Regulation of Gabaergic and Glutamatergic Pathways

To identify genetic variants associated with greater tobacco consumption in a Mexican population. Daily smokers were classified as light smokers (LS; n = 742), heavy smokers (HS; n = 601) and nonsmokers (NS; n = 606). In the first stage, a genotyping microarray that included 347 SNPs in CHRNA2-CHRNA7/CHRNA10, CHRNB2-CHRNB4 and NRXN1 genes and 37 ancestry-informative markers was used to analyze 707 samples (187 HS, 328 LS and 192 NS). In the second stage, 14 SNPs from stage 1 were validated in the remaining samples (HS, LS and NS; n = 414 in each group) using real-time PCR. To predict the role of the associated SNPs, an in silico analysis was performed. Two SNPs in NRXN1 and two in CHRNA5 were associated with cigarette consumption, while rs10865246/C (NRXN1) was associated with high nicotine addiction. The in silico analysis revealed that rs1882296/T had a high level of homology with Hsa-miR-6740-5p, which encodes a putative miRNA that targets glutamate receptor subunits (GRIA2, GRID2) and GABA receptor subunits (GABRG1, GABRA4, GABRB2), while rs1882296/C had a high level of homology with Hsa-miR-6866-5p, which encodes a different miRNA that targets GRID2 and GABRB2. In a Mexican Mestizo population, greater consumption of cigarettes was influenced by polymorphisms in the NRXN1 and CHRNA5 genes. We proposed new hypotheses regarding the putative roles of miRNAs that influence the GABAergic and glutamatergic pathways in smoking addiction.