Putative miRNA Research Articles

MiR156a Mimic Represses the Epithelial–Mesenchymal Transition of Human Nasopharyngeal Cancer Cells by Targeting Junctional Adhesion Molecule A

MicroRNAs (miRNAs) have been documented as having an important role in the development of cancer. Broccoli is very popular in large groups of the population and has anticancer properties. Junctional adhesion molecule A (JAMA) is preferentially concentrated at tight junctions and influences cell morphology and migration. Epithelial–mesenchymal transition (EMT) is a developmental program associated with cancer progression and metastasis. In this study we aimed to investigate the role of miRNAs from broccoli in human nasopharyngeal cancer (NPC). We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology. Among these, miR156a was expressed the most. In addition, synthetic miR156a mimic inhibited the EMT of NPC cells in vitro. Furthermore, it was confirmed that JAMA was the target of miR156a mimic as validated by 3’ UTR luciferase reporter assays and western blotting. Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic. In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3’ UTR of JAMA. These miRNA profiles of broccoli provide a fundamental basis for further research. Moreover, the discovery of miR156a may have clinical implications for the treatment of patients with NPC.

Ibrutinib downregulates a subset of miRNA leading to upregulation of tumor suppressors and inhibition of cell proliferation in chronic lymphocytic leukemia.

The lymph node (LN) is the site of chronic lymphocytic leukemia (CLL) cell activation and proliferation. Aberrant microRNA (miRNA) expression has been shown to have a role in CLL pathogenesis; however, a comparison of miRNA expression between CLL cells in the LN and the peripheral blood (PB) has previously not been reported. On the basis of the analysis of 17 paired LN and PB samples from CLL patients, we identify a panel of miRNAs that are increased in LN CLL cells correlating with an activation phenotype. When evaluated in CLL cells from 38 patients pre and post treatment with ibrutinib, a subset of these miRNAs (miR-22, miR-34a, miR-146b and miR-181b) was significantly decreased in response to ibrutinib. A concomitant increase in putative miRNA target transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) was also observed. Functional studies confirmed targets of ibrutinib-responsive miRNAs to include messenger RNA transcripts of multiple tumor suppressors. Knockdown of endogenous miR-34a and miR146b resulted in increased transcription of tumor suppressors and inhibition of cell proliferation. These findings demonstrate that ibrutinib downregulates the expression of a subset of miRNAs related to B-cell activation leading to increased expression of miRNA targets including tumor suppressors and a reduction in cell proliferation.

Identification of microRNAs and their targets in Paulownia fortunei plants free from phytoplasma pathogen after methyl methane sulfonate treatment.

MicroRNAs (miRNAs) play major roles in plant responses to various biotic and abiotic stresses by regulating gene expression at the transcriptional and post-transcriptional levels. Paulownia witches' broom (PaWB) disease caused by phytoplasmas reduces Paulownia production worldwide. In this study, we investigated the miRNA-mediated plant response to PaWB phytoplasma by Illumina sequencing and degradome analysis of Paulownia fortunei small RNAs (sRNAs). The sRNA and degradome libraries were constructed from healthy and diseased P.fortunei plants and the plants free from phytoplasma pathogen after 60mgL(-1) methyl methane sulfonate treatment. A total of 96 P.fortunei-conserved miRNAs and 83 putative novel miRNAs were identified. Among them, 37 miRNAs (17 conserved, 20 novel) were found to be differentially expressed in response to PaWB phytoplasma infection. In addition, 114 target genes for 18 of the conserved miRNA families and 33 target genes for 15 of the novel miRNAs in P.fortunei were detected. The expression patterns of 14 of the PaWB phytoplasma-responsive miRNAs and 12 target genes were determined by quantitative real-time polymerase chain reaction (qPCR) experiments. A functional analysis of the miRNA targets indicated that these targeted genes may regulate transcription, stress response, nitrogen metabolism, and various other activities. Our results will help identify the potential roles of miRNAs involved in protecting P.fortunei from diseases.

A naïve Bayesian classifier for identifying plant microRNAs

MicroRNAs (miRNAs) are important regulatory molecules in eukaryotic organisms. Existing methods for the identification of mature miRNA sequences in plants rely extensively on the search for stem-loop structures, leading to high false negative rates. Here, we describe a probabilistic method for ranking putative plant miRNAs using a naïve Bayes classifier and its publicly available implementation. We use a number of properties to construct the classifier, including sequence length, number of observations, existence of detectable predicted miRNA* sequences, the distribution of nearby reads and mapping multiplicity. We apply the method to small RNA sequence data from soybean, peach, Arabidopsis and rice and provide experimental validation of several predictions in soybean. The approach performs well overall and strongly enriches for known miRNAs over other types of sequences. By utilizing a Bayesian approach to rank putative miRNAs, our method is able to score miRNAs that would be eliminated by other methods, such as those that have low counts or lack detectable miRNA* sequences. As a result, we are able to detect several soybean miRNA candidates, including some that are 24 nucleotides long, a class that is almost universally eliminated by other methods.

Association of MiR-26b mediated doxorubicin resistance signature with poor prognosis in epithelial ovarian cancer (EOC).

e17070Background: MicroRNAs (miRNA) have been established as key regulators of tumor gene expression as well as prime biomarker candidates for clinical phenotypes. We analyzed the regulatory structure of miRNAs and their colocalized gene targets in EOC tissue to construct a regulatory signature for clinical prognosis. Methods: FFPE blocks of primary EOC tumor tissue with > 80% tumor cells were selected for 20 patients (stage ≥ IIc) for whom follow up data was available. Expression levels of mRNA and miRNA were assayed separately. An integrated analysis was performed to identify miRNA/mRNA regulatory clusters. Gene set enrichment analysis (GSEA) was carried out on the coexpression of putative miRNA target genes, from the miRTarBase database, with their respective miRNAs, to identify enriched categories for each miRNA. MiRNAs with at least one significantly enriched category from the MSigDB C2 dataset at a false discovery rate of 0.05 were chosen. For each enriched category of each miRNA, a regulon was defi...

Dissecting miRNAs in Wheat D Genome Progenitor, Aegilops tauschii.

As the post-transcriptional regulators of gene expression, microRNAs or miRNAs comprise an integral part of understanding how genomes function. Although miRNAs have been a major focus of recent efforts, miRNA research is still in its infancy in most plant species. Aegilops tauschii, the D genome progenitor of bread wheat, is a wild diploid grass exhibiting remarkable population diversity. Due to the direct ancestry and the diverse gene pool, A. tauschii is a promising source for bread wheat improvement. In this study, a total of 87 Aegilops miRNA families, including 51 previously unknown, were computationally identified both at the subgenomic level, using flow-sorted A. tauschii 5D chromosome, and at the whole genome level. Predictions at the genomic and subgenomic levels suggested A. tauschii 5D chromosome as rich in pre-miRNAs that are highly associated with Class II DNA transposons. In order to gain insights into miRNA evolution, putative 5D chromosome miRNAs were compared to its modern ortholog, Triticum aestivum 5D chromosome, revealing that 48 of the 58 A. tauschii 5D miRNAs were conserved in orthologous T. aestivum 5D chromosome. The expression profiles of selected miRNAs (miR167, miR5205, miR5175, miR5523) provided the first experimental evidence for miR5175, miR5205 and miR5523, and revealed differential expressional changes in response to drought in different genetic backgrounds for miR167 and miR5175. Interestingly, while miR5523 coding regions were present and expressed as pre-miR5523 in both T. aestivum and A. tauschii, the expression of mature miR5523 was observed only in A. tauschii under normal conditions, pointing out to an interference at the downstream processing of pre-miR5523 in T. aestivum. Overall, this study expands our knowledge on the miRNA catalog of A. tauschii, locating a subset specifically to the 5D chromosome, with ample functional and comparative insight which should contribute to and complement efforts to develop drought tolerant wheat varieties.

A Series of microRNA in the Chromosome 14q32.2 Maternally Imprinted Region Related to Progression of Non-Alcoholic Fatty Liver Disease in a Mouse Model.

Background & AimsSimple steatosis (SS) and non-alcoholic steatohepatitis (NASH) are subtypes of non-alcoholic fatty liver disease (NAFLD), and the pathogenic differences between SS and NASH remain unclear. MicroRNAs (miRNAs) are endogenous, non-coding, short RNAs that regulate gene expression. The aim of this study was to use animal models and human samples to examine the relationship between miRNA expression profiles and each type of NAFLD (SS and NASH).MethodsDD Shionogi, Fatty Liver Shionogi (FLS) and FLS ob/ob mice were used as models for normal control, SS and NASH, respectively. Microarray analysis and real-time PCR were used to identify candidate NAFLD-related miRNAs. Human serum samples were used to examine the expression profiles of these candidate miRNAs in control subjects and patients with SS or NASH.ResultsFourteen miRNAs showed clear expression differences among liver tissues from SS, NASH, and control mice with good reproducibility. Among these NAFLD candidate miRNAs, seven showed similar expression patterns and were upregulated in both SS and NASH tissues; these seven candidate miRNAs mapped to an miRNA cluster in the 14q32.2 maternally imprinted region delineated by delta-like homolog 1 and type III iodothyronine deiodinase (Dlk1-Dio3 mat). Software-based predictions indicated that the transforming growth factor-β pathway, insulin like growth factor-1 and 5' adenosine monophosphate activated protein kinase were potential targets of theses Dlk1-Dio3 mat NAFLD candidate miRNAs. In addition, serum samples from patients with SS or NASH differed markedly with regard to expression of the putative Dlk1-Dio3 mat miRNAs, and these differences accurately corresponded with NAFLD diagnosis.ConclusionThe expression profiles of seven miRNAs in 14q32.2 mat have high potential as biomarkers for NAFLD and for improving future research on the pathogenesis and treatment of NASH.

Identification of novel stress-induced microRNAs and their targets in Camelina sativa using computational approach

MicroRNAs (miRNAs) are a class of small non-coding, single-stranded RNA sequences that regulate gene expression at the post-transcriptional level and also reported to function in stress responses, but their role has not been studied in Camelina (Camelina sativa L.), an emerging oil crop. In this study, we predicted conserved as well as putative novel miRNAs from a Camelina drought stress cDNA library using comprehensive genomic approaches. Based on the sequence homology, we predicted 145 miRNAs, of which 61 were conserved, and 84 putative novel miRNAs were found to belong to 26 and 72 different miRNA families, respectively. In silico expression analysis indicated that 20 miRNAs were really expressed in Camelina genome, and several of them have tissue-specific expression character. We found that the 60 putative novel miRNA families target 117 genes. Most of the miRNA targets were predicted to genes including that regulate stress response, transcription factors, and fatty acid and lipid metabolism-related genes. Expression patterns of 6 randomly selected miRNAs under drought stress were validated by real-time quantitative polymerase chain reaction analysis. Coordinated expression changes between 6 randomly selected miRNAs and their target genes, suggested that the predicted miRNAs could be drought-responsive and that they would likely be directly involved in stress regulatory networks of Camelina. These results indicate that, in C. sativa, under drought stress, a large number of new miRNAs could be discovered, and the predicted stress-responsive miRNAs and their target transcripts will serve as valuable resources for future studies.

MiR-34a-5p Inhibition Alleviates Intestinal Ischemia/Reperfusion-Induced Reactive Oxygen Species Accumulation and Apoptosis via Activation of SIRT1 Signaling.

Reactive oxygen species (ROS) generation and massive epithelial apoptosis are critical in the pathogenesis of intestinal ischemia/reperfusion (I/R) injury. We previously found that the Sirtuin 1 (SIRT1)-mediated antioxidant pathway was impaired in the intestine after I/R. Here, we investigate the potential role of SIRT1-targeting microRNAs (miRNAs) in regulating ROS accumulation and apoptosis in intestinal I/R, and the important role SIRT1 involved in. C57BL/6 mice were subjected to intestinal I/R induced by occlusion of the superior mesenteric artery followed by reperfusion. Caco-2 cells were incubated under hypoxia/reoxygenation condition to mimic I/R in vivo. We find that SIRT1 is gradually repressed during the early reperfusion, and that this repression results in intestinal ROS accumulation and apoptosis. Using bioinformatics analysis and real-time PCR, we demonstrate that miR-34a-5p and miR-495-3p are significantly increased among the 41 putative miRNAs that can target SIRT1. Inhibition of miR-34a-5p, but not miR-495-3p, attenuates intestinal I/R injury, as demonstrated by repressing p66shc upregulation, manganese superoxide dismutase repression, and the caspase-3 activation in vitro and in vivo; it further alleviates systemic injury, as demonstrated by reducing inflammatory cytokine release, attenuating lung and liver lesions, and improving survival. Interestingly, SIRT1 plays an indispensable role in the protection afforded by miR-34a-5p inhibition. This study provides the first evidence of miRNAs in regulating oxidative stress and apoptosis in intestinal I/R. miR-34a-5p knockdown attenuates intestinal I/R injury through promoting SIRT1-mediated suppression of epithelial ROS accumulation and apoptosis. This may represent a novel prophylactic approach to intestinal I/R injury. Antioxid. Redox Signal. 24, 961-973.

Deep-sequence profiling of miRNAs and their target prediction in Monotropa hypopitys.

Myco-heterotroph Monotropa hypopitys is a widely spread perennial herb used to study symbiotic interactions and physiological mechanisms underlying the development of non-photosynthetic plant. Here, we performed, for the first time, transcriptome-wide characterization of M. hypopitys miRNA profile using high throughput Illumina sequencing. As a result of small RNA library sequencing and bioinformatic analysis, we identified 55 members belonging to 40 families of known miRNAs and 17 putative novel miRNAs unique for M. hypopitys. Computational screening revealed 206 potential mRNA targets for known miRNAs and 31 potential mRNA targets for novel miRNAs. The predicted target genes were described in Gene Ontology terms and were found to be involved in a broad range of metabolic and regulatory pathways. The identification of novel M. hypopitys-specific miRNAs, some with few target genes and low abundances, suggests their recent evolutionary origin and participation in highly specialized regulatory mechanisms fundamental for non-photosynthetic biology of M. hypopitys. This global analysis of miRNAs and their potential targets in M. hypopitys provides a framework for further investigation of miRNA role in the evolution and establishment of non-photosynthetic myco-heterotrophs.

Peripheral whole blood microRNA alterations in major depression and bipolar disorder

Major depression (MD) and bipolar disorder (BD) are severe and potentially life-threating mood disorders whose etiology is to date not completely understood. MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein synthesis post-transcriptionally by base-pairing to target gene mRNAs. Growing evidence indicated that miRNAs might play a key role in the pathogenesis of neuropsychiatric disorders and in the action of psychotropic drugs. On these bases, in this study we evaluated the expression levels of 1733 mature miRNAs annotated in miRBase v.17, through a microarray technique, in the blood of 20 MD and 20 BD patients and 20 healthy controls, in order to identify putative miRNA signatures associated with mood disorders. We found that 5 miRNAs (hsa-let-7a-5p, hsa-let-7d-5p, hsa-let-7f-5p, hsa-miR-24-3p and hsa-miR-425-3p) were specifically altered in MD patients and 5 (hsa-miR-140-3p, hsa-miR-30d-5p, hsa-miR-330-5p, hsa-miR-378a-5p and hsa-miR-21-3p) in BD patients, whereas 2 miRNAs (hsa-miR-330-3p and hsa-miR-345-5p) were dysregulated in both the diseases. The bioinformatic prediction of the genes targeted by the altered miRNAs revealed the possible involvement of neural pathways relevant for psychiatric disorders. In conclusion, the observed results indicate a dysregulation of miRNA blood expression in mood disorders and could indicate new avenues for a better understanding of their pathogenetic mechanisms. The identified alterations may represent potential peripheral biomarkers to be complemented with other clinical and biological features for the improvement of diagnostic accuracy.

Alcohol Use During Pregnancy is Associated with Specific Alterations in MicroRNA Levels in Maternal Serum.

BackgroundGiven the challenges of confirming prenatal alcohol exposure (PAE) during pregnancy using currently established biomarkers of alcohol consumption, we examined whether serum microRNAs (miRNAs) may serve as stable biomarkers for PAE. Alterations in the levels of specific circulating miRNAs have been associated with various disease states and in animal models of fetal alcohol spectrum disorder.MethodsPregnant women in this prospective study were recruited from substance abuse and general maternity clinics affiliated with the University of New Mexico. Serum was collected at the time of admission for delivery from 14 subjects who reported ≥1 binge‐drinking episode or ≥3 drinks/wk during pregnancy and 16 subjects who reported abstinence during pregnancy and tested negative for 5 ethanol biomarkers. Total RNA was isolated from serum and used for microarray analysis.ResultsFalse discovery rate‐corrected analyses of covariance revealed that 55 miRNAs were significantly altered between the 2 groups. Hierarchical clustering using only the significantly altered miRNAs grouped samples into alcohol‐consuming and non‐alcohol‐consuming individuals. Discriminant analysis then identified miRs‐122*, ‐126, ‐216b, ‐221*, ‐3119, ‐3942‐5p, ‐4704‐3p, ‐4743, ‐514‐5p, and ‐602 as the top 10 discriminators between the 2 groups. Ingenuity Pathway Analysis of putative miRNA targets illustrated that miRNAs identified in this study are involved in biological pathways that mediate the effects of alcohol, such as brain‐derived neurotrophic factor, ERK1/2, and PI3K/AKT signaling.ConclusionsThis is the first report of alterations in serum miRNA expression that are associated with alcohol use during human pregnancy. These results suggest that serum miRNAs could be useful as biomarkers of alcohol exposure.

Expression profile of androgen-modulated microRNAs in the fetal murine lung.

BackgroundAndrogens are known to delay lung development. As a consequence, the incidence and morbidity of respiratory distress syndrome of the neonate are higher for male than for female premature infants. We previously reported that many genes were expressed with a sex difference in the mouse developing lung and that several genes were under the control of androgens in the male fetal lung. microRNAs are small non-coding RNAs known to negatively regulate the expression of specific genes. In this study, we examined whether murine miRNAs are under the control of androgens in the male developing lung.MethodsExpression profiling of microRNAs was performed by microarrays using RNA extracted from male fetal lungs isolated on gestational day (GD) 17.0 and GD 18.0 after daily injection of pregnant mice from GD 10.0 with the antiandrogen flutamide or vehicle only. To identify putative miRNA target genes, the data obtained here were combined with gene profiling data reported previously using the same RNA preparations. qPCR was used to confirm microarray data with fetal lungs from other litters than those used in microarrays.ResultsFlutamide induced downregulation and upregulation of several miRNAs on GD 17.0 and GD 18.0. Of the 43 mature miRNAs modulated by flutamide on GD 17.0, 60 % were downregulated, whereas this proportion was only of 34 % for the 35 mature miRNAs modulated on GD 18.0. For 29 and 26 flutamide-responsive miRNAs, we found a corresponding target inversely regulated by androgens on GD 17.0 and 18.0, respectively. The androgen-regulated target genes were involved in several biological processes (lipid metabolism, cell proliferation, and lung development) and molecular functions, mainly transcription factor binding.ConclusionsRegulation of male lung development involves several miRNAs that are under androgen modulation in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-016-0072-z) contains supplementary material, which is available to authorized users.

Characterization of microRNAs and their targets in wild barley (Hordeum vulgare subsp. spontaneum) using deep sequencing.

MicroRNAs (miRNA) are a class of small, endogenous RNAs that play a negative regulatory role in various developmental and metabolic processes of plants. Wild barley (Hordeum vulgare subsp. spontaneum), as the progenitor of cultivated barley (Hordeum vulgare subsp. vulgare), has served as a valuable germplasm resource for barley genetic improvement. To survey miRNAs in wild barley, we sequenced the small RNA library prepared from wild barley using the Illumina deep sequencing technology. A total of 70 known miRNAs and 18 putative novel miRNAs were identified. Sequence analysis revealed that all of the miRNAs identified in wild barley contained the highly conserved hairpin sequences found in barley cultivars. MiRNA target predictions showed that 12 out of 52 miRNA families were predicted to target transcription factors, including 8 highly conserved miRNA families in plants and 4 wheat-barley conserved miRNA families. In addition to transcription factors, other predicted target genes were involved in diverse physiological and metabolic processes and stress defense. Our study for the first time reported the large-scale investigation of small RNAs in wild barley, which will provide essential information for understanding the regulatory role of miRNAs in wild barley and also shed light on future practical utilization of miRNAs for barley improvement.

Stromal Expression of miR-143/145 Promotes Neoangiogenesis in Lung Cancer Development.

The two unrelated miRNAs miR-143 and miR-145, coexpressed from the miR-143/145 cluster, have been proposed to act as tumor suppressors in human cancer, and therapeutic benefits of delivering miR-143 and miR-145 to tumors have been reported. In contrast, we found that tumor-specific deletion of miR-143/145 in an autochthonous mouse model of lung adenocarcinoma did not affect tumor development. This was consistent with the lack of endogenous miR-143/145 expression in normal and transformed lung epithelium. Surprisingly, miR-143/145 in the tumor microenvironment dramatically promoted tumor growth by stimulating the proliferation of endothelial cells. Loss of miR-143/145 in vivo led to derepression of the miR-145 target CAMK1D, an inhibitory kinase, which when overexpressed prevents mitotic entry of endothelial cells. As a consequence, tumors in miR-143/145-deficient animals exhibited diminished neoangiogenesis, increased apoptosis, and their expansion was limited by the tumor's ability to co-opt the alveolar vasculature. These findings demonstrate that stromal miR-143/145 promotes tumorigenesis and caution against the use of these miRNAs as agents in cancer therapeutics. This study shows that miR-143/145 expressed from the tumor microenvironment stimulates neoangiogenesis and supports tumor expansion in the lung, demonstrating a surprising role for the putative tumor suppressor miRNA cluster in promoting tumorigenesis. We propose inhibition of miR-143/145 as a therapeutic avenue to modulate tumor neoangiogenesis.

Identification and validation of a virus-inducible ta-siRNA-generating TAS4 locus in tomato.

Trans-acting small interfering RNAs (ta-siRNAs) are a class of endogenous small RNA, associated with posttranscriptional gene silencing. Their biogenesis requires an initial microRNA (miRNA)-mediated cleavage of precursor RNA. Around 20 different ta-siRNA-producing loci (TASs), whose sequences are conserved, are reported in plants. In tomato, two TAS gene families have been identified, which are found to target auxin response factor gene and bacterial spot disease resistance protein Bs4 gene. Using high-throughput computational and experimental approach, we identified a new locus-producing ta-siRNA in tomato. We have also identified the putative miRNA regulating the production of ta-siRNA from this locus. The ta-siRNAs generated from TAS4 were up-regulated upon infection with a DNA virus. The potential targets of ta-siRNAs were predicted to be variety of proteins including MYB transcription factors and cell cycle regulators for some of the ta-siRNAs produced.

Unveiling the Micronome of Cassava (Manihot esculenta Crantz).

MicroRNAs (miRNAs) are an important class of endogenous non-coding single-stranded small RNAs (21–24 nt in length), which serve as post-transcriptional negative regulators of gene expression in plants. Despite the economic importance of Manihot esculenta Crantz (cassava) only 153 putative cassava miRNAs (from multiple germplasm) are available to date in miRBase (Version 21), and identification of a number of miRNAs from the cassava EST database have been limited to comparisons with Arabidopsis. In this study, mature sequences of all known plant miRNAs were used as a query for homologous searches against cassava EST and GSS databases, and additional identification of novel and conserved miRNAs were gleaned from next generation sequencing (NGS) of two cassava landraces (T200 from southern Africa and TME3 from West Africa) at three different stages post explant transplantation and acclimatization. EST and GSS derived data revealed 259 and 32 miRNAs in cassava, and one of the miRNA families (miR2118) from previous studies has not been reported in cassava. NGS data collectively displayed expression of 289 conserved miRNAs in leaf tissue, of which 230 had not been reported previously. Of the 289 conserved miRNAs identified in T200 and TME3, 208 were isomiRs. Thirty-nine novel cassava-specific miRNAs of low abundance, belonging to 29 families, were identified. Thirty-eight (98.6%) of the putative new miRNAs identified by NGS have not been previously reported in cassava. Several miRNA targets were identified in T200 and TME3, highlighting differential temporal miRNA expression between the two cassava landraces. This study contributes to the expanding knowledge base of the micronome of this important crop.

Identification and Expression Analyses of miRNAs from Two Contrasting Flower Color Cultivars of Canna by Deep Sequencing

miRNAs are endogenous small RNA (sRNA) that play critical roles in plant development processes. Canna is an ornamental plant belonging to family Cannaceae. Here, we report for the first time the identification and differential expression of miRNAs in two contrasting flower color cultivars of Canna, Tropical sunrise and Red president. A total of 313 known miRNAs belonging to 78 miRNA families were identified from both the cultivars. Thirty one miRNAs (17 miRNA families) were specific to Tropical sunrise and 43 miRNAs (10 miRNA families) were specific to Red president. Thirty two and 18 putative new miRNAs were identified from Tropical sunrise and Red president, respectively. One hundred and nine miRNAs were differentially expressed in the two cultivars targeting 1343 genes. Among these, 16 miRNAs families targeting60 genes were involved in flower development related traits and five miRNA families targeting five genes were involved in phenyl propanoid and pigment metabolic processes. We further validated the expression analysis of a few miRNA and their target genes by qRT-PCR. Transcription factors were the major miRNA targets identified. Target validation of a few randomly selected miRNAs by RLM-RACE was performed but was successful with only miR162. These findings will help in understanding flower development processes, particularly the color development in Canna.

Circulating microRNAs as Potential Biomarkers of Endothelial Dysfunction in Obese Children.

Cardiovascular disease (CVD) is a complex disease with multifactorial etiology. The presence of endothelial dysfunction constitutes an early risk factor for CVD in children. Circulating microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and represent a novel class of biomarkers and therapeutic targets; therefore, we examined whether the presence of endothelial dysfunction is associated with differential expression of plasma miRNAs in otherwise healthy children. A total of 70 children (aged 5-10 years) were recruited and classified into two groups (normal endothelial function [NEF] and endothelial dysfunction). Time to peak postocclusive reperfusion (Tmax) was considered as the indicator of either normal endothelial function (NEF; Tmax < 45 s) or endothelial dysfunction (Tmax ≥ 45 s). Lipid profiles, high-sensitivity C-reactive protein, fasting glucose, and insulin were assayed using enzyme-linked immunosorbent assay. miRNAs isolated from plasma were assayed with a custom human CVD array, followed by quantitative polymerase chain reaction verification of candidates. In addition, bioinformatics approaches including combinatorial target prediction algorithms and gene ontology were applied. Three miRNAs that have been previously linked to cardiomyopathy, hsa-miR-125a-5p, hsa-miR-342-3p, and hsa-miR-365b-3p, were identified as potential biomarkers of children with endothelial dysfunction. The miRNA predicted gene targets revealed 31 common targets among all three putative candidate biomarker miRNAs and encompass three biologic pathways, including transforming growth factor-β signaling, cytokine-cytokine receptor interactions, and activin receptor-like kinase in cardiac myocytes. Plasma miRNAs may be useful as potential screening tools for the presence of endothelial dysfunction in children and may reveal endothelial dysfunction-relevant target genes.

Multiple microRNA regulation of lipoprotein lipase gene abolished by 3′UTR polymorphisms in a triglyceride-lowering haplotype harboring p.Ser474Ter

BackgroundLipoprotein lipase (LPL) is a key enzyme in triglyceride (TG) metabolism. LPL gene single nucleotide polymorphisms (SNPs) are associated with TG concentrations however the functionality of many of these SNPs remains poorly understood. MicroRNAs (miR) exert post-transcriptional down-regulation and their target sequence on the 3′UTR may be altered by SNPs. We therefore investigated whether LPL 3′UTR SNPs could modulate plasma TG concentration through the alteration of miR binding-sites. Methods and resultsWe performed genetic association studies of LPL 3′UTR SNPs with TG concentrations in 271 type 2 diabetic patients and in general population samples (2997 individuals). A specific LPL haplotype (Hap4) was associated with lower plasma TG concentration (TG-0.18, IC95% [−0.30, −0.07] mmol/L or logTG-0.13, IC95% [−0.18, −0.08], p = 4.77·10−8) in the meta-analysis. Hap4 comprises seven 3′UTR SNP minor alleles and p.Ser474Ter (rs328) a well-documented nonsense mutation associated with low TG concentration although by an unknown mechanism so far. Bio-informatic studies identified several putative miRNA binding-sites on the wild-type Hap1 haplotype, lost on Hap4. Functional validation performed in HEK-293T cells using luciferase expression constructs with various LPL 3′UTR allele combinations demonstrated a binding of miR-29, miR-1277 and miR-410 on Hap1, lost on Hap4. This loss of specific miR binding-site in presence of Hap4 was independent of the allelic variation of p.Ser474Ter (rs328). ConclusionsWe report the regulation of LPL by the miR-29, miR-1277 and miR-410 that is lost in presence of Hap4, a specific LPL TG-lowering haplotype. Consequently p.Ser474Ter association with TG concentration could be at least partially explained by its strong linkage disequilibrium with these functional 3′UTR SNPs.