Mature miRNA Sequences Research Articles

The role of variations within microRNA in inflammatory bowel disease

MicroRNAs (miRNAs) are small noncoding RNAs that are implicated in gene expression regulation at both a transcriptional and at a translational level. Single-nucleotide polymorphisms may occur in miRNA biogenesis pathway genes, primary miRNA, pre-miRNA, or a mature miRNA sequence. Such polymorphisms may be functional with respect to biogenesis and actions of mature miRNA. These single-nucleotide polymorphisms may have a potential to affect the efficiency of miRNA binding to the target sites or can create or disrupt binding sites. The resulting gene dysregulation may involve changes in phenotype and may eventually prove critical for the susceptibility to inflammatory bowel disease and its onset. In this review, we summarize their importance as candidate inflammatory bowel disease biomarkers.

Global profiling of miRNAs and the hairpin precursors: insights into miRNA processing and novel miRNA discovery

MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5′ tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences.

The Sequence Structures of Human MicroRNA Molecules and Their Implications

The count of the nucleotides in a cloned, short genomic sequence has become an important criterion to annotate such a sequence as a miRNA molecule. While the majority of human mature miRNA sequences consist of 22 nucleotides, there exists discrepancy in the characteristic lengths of the miRNA sequences. There is also a lack of systematic studies on such length distribution and on the biological factors that are related to or may affect this length. In this paper, we intend to fill this gap by investigating the sequence structure of human miRNA molecules using statistics tools. We demonstrate that the traditional discrete probability distributions do not model the length distribution of the human mature miRNAs well, and we obtain the statistical distribution model with a decent fit. We observe that the four nucleotide bases in a miRNA sequence are not randomly distributed, implying that possible structural patterns such as dinucleotide (trinucleotide or higher order) may exist. Furthermore, we study the relationships of this length distribution to multiple important factors such as evolutionary conservation, tumorigenesis, the length of precursor loop structures, and the number of predicted targets. The association between the miRNA sequence length and the distributions of target site counts in corresponding predicted genes is also presented. This study results in several novel findings worthy of further investigation that include: (1) rapid evolution introduces variation to the miRNA sequence length distribution; (2) miRNAs with extreme sequence lengths are unlikely to be cancer-related; and (3) the miRNA sequence length is positively correlated to the precursor length and the number of predicted target genes.

Simultaneous Detection of Different MicroRNA Types Using the ZIP-Code Array System

MicroRNAs (miRNAs) are important negative regulators of gene expression. Their implication in tumorigenesis is based on their dysregulation in many human cancer diseases. Interestingly, in tumor cells, an altered ratio of precursor and mature miRNA levels has been described. Consequently, differences in miRNA type levels have a high potential as biomarkers and comparative high-throughput-based detection might permit a more accurate characterization of subtypes, especially in the case of very heterogeneous tumor entities. Several molecular methods exist for the detection of mature and precursor miRNAs. DNA microarrays are predestinated as a high-throughput method for comprehensive miRNA detection in tumors. However, the simultaneous array-based detection of both these miRNA types is limited because the mature miRNA sequence is identically present in both forms. Here we present a ZIP-code DNA microarray-based system in combination with a novel labeling approach, which enables the simultaneous detection of precursor and mature miRNAs in one single experiment. Using synthetic miRNA templates, we demonstrate the specificity of the method for the different miRNA types, as well as the detection range up to four orders of magnitude. Moreover, mature and precursor miRNAs were detected and validated in human tumor cells.

Biocomputational genome-wide analysis of micro RNA genetic variability in some vertebrates

MicroRNAs (miRNAs) are small endogenously expressed single-stranded RNAs that regulate gene expression post transcriptionally and shape diverse cellular pathways. miRNAs regulate a wide range of biological processes through the recognition of complementary sequences between miRNAs and their target genes. The present investigation aimed at determining in-silico the genetic variability of miRNA genes in some livestock and non-livestock species. Effects of single nucleotide polymorphisms (SNPs) in genes? 3'UTR on target gain/loss of human miRNAs were also explored. A total of twenty four mature miRNA sequences and genomic coordinates in three livestock [chicken (5), pig (1) and cattle (9)] and two non-livestock (human (6) and mouse (3)] species were retrieved from the miRBase 15 release. Computational scanning of polymorphisms in the miRNAs revealed 33 and 20 polymorphic sites in livestock and non-livestock species, respectively. Of this, 7 (chicken), 11 (cattle) and 2 (mouse) were located within the seed region. The de novo computational prediction revealed that SNPs rs1042725 (C/U) and rs1044129 (A/G) in genes? 3'UTR of human miRNAs positively influenced the target site thereby resulting in target gain. However, the effects of SNPs rs56109847 (A/G), rs28927680, rs12720208 (G/A) and rs5186 (A/C) were negative. The evolutionary tree showed that the relationship between miRNA consensus sequences of livestock (pig, chicken and cattle) was closer compared to non-livestock species (mouse and human), which could be implicated in morphological complexity among vertebrates. Although the function of miRNA is only beginning to be understood, future in-silico research evaluating the functional effect of miRNA in gene translation and subsequent biological pathways especially in livestock is of paramount importance; and this should be complemented with hypothesis-driven experimental studies to evaluate the phenotypic effect of identified miRNA genetic polymorphisms in animals.

MicroRNA expression profiles of LO2 cells expressing the wild-type and mutant HBx gene

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although numerous studies have suggested the potentially oncogenic roles of wild‑type or mutant hepatitis B virus X (HBx) protein in hepatocarcinogenesis, their exact mechanism remains unclear. Increasing evidence suggests that microRNAs (miRNAs) play essential roles in embryogenesis, cell differentiation and carcinogenesis. This study aimed to investigate the effect of HBx on the miRNA expression profile of LO2 cells. We established the LO2 cell line transfected with recombinant plasmid pcDNA3.0/HBx-d382, pcDNA/HBx and plasmid pcDNA3.0 using Lipofectamine™ 2000, which was confirmed by reverse transcription‑polymerase chain reaction (RT-PCR) and western blotting. We then demonstrated the miRNA expression profiles in the stably transfected LO2 cells using a mammalian miRNA microarray containing whole‑human mature and precursor miRNA sequences. The results were confirmed by real-time quantitative PCR (qPCR). RT-PCR and western blot analysis showed that a stably HBx‑transfected LO2 cell line had been successfully established. According to the microarray, compared to LO2/pcDNA3.0 cells, 6 miRNAs were shown to have higher expression and 5 were shown to have decreased expression in LO2/HBx-d382 cells, while 4 up- and 12 downregulated miRNAs were observed in LO2/HBx cells. There were 8 different expression patterns of miRNAs between LO2/HBx and LO2/HBx-d382 cells. All the chip results were consistent with the real‑time PCR data. Consequently, the HBx gene may influence the miRNA expression profile of LO2 cells. Thus, it may be helpful to further investigate the role of HBx in hepatocarcinogenesis and clarify the underlying molecular mechanisms involved.

Identification of MicroRNAs from Eugenia uniflora by High-Throughput Sequencing and Bioinformatics Analysis

BackgroundmicroRNAs or miRNAs are small non-coding regulatory RNAs that play important functions in the regulation of gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. Eugenia uniflora is a plant native to tropical America with pharmacological and ecological importance, and there have been no previous studies concerning its gene expression and regulation. To date, no miRNAs have been reported in Myrtaceae species.ResultsSmall RNA and RNA-seq libraries were constructed to identify miRNAs and pre-miRNAs in Eugenia uniflora. Solexa technology was used to perform high throughput sequencing of the library, and the data obtained were analyzed using bioinformatics tools. From 14,489,131 small RNA clean reads, we obtained 1,852,722 mature miRNA sequences representing 45 conserved families that have been identified in other plant species. Further analysis using contigs assembled from RNA-seq allowed the prediction of secondary structures of 25 known and 17 novel pre-miRNAs. The expression of twenty-seven identified miRNAs was also validated using RT-PCR assays. Potential targets were predicted for the most abundant mature miRNAs in the identified pre-miRNAs based on sequence homology.ConclusionsThis study is the first large scale identification of miRNAs and their potential targets from a species of the Myrtaceae family without genomic sequence resources. Our study provides more information about the evolutionary conservation of the regulatory network of miRNAs in plants and highlights species-specific miRNAs.

Effects of drought on the microtranscriptome of field-grown sugarcane plants

Sugarcane (Saccharum spp.) is the most promising crop for renewable energy. Among the diverse stresses that affect plant productivity, drought stress frequently causes losses in sugarcane fields. Although several studies have addressed plant responses to drought using controlled environments, plant responses under field conditions are largely unknown. Recently, microRNA (miRNA)-mediated post-transcriptional regulation has been described as an important and decisive component in vegetal development and stress resistance modulation. The role of miRNAs in sugarcane responses to drought under field conditions is currently not known. Two sugarcane cultivars differing in drought tolerance were grown in the field with and without irrigation (rainfed) for 7 months. By using small RNA deep sequencing, we were able to identify 18 miRNA families comprising 30 mature miRNA sequences. Among these families, we found 13 mature miRNAs that were differentially expressed in drought-stressed plants. Seven miRNAs were differentially expressed in both cultivars. The target genes for many of the differentially expressed mature miRNAs were predicted, and some of them were validated by quantitative reverse transcription PCR. Among the targets, we found transcription factors, transporters, proteins associated with senescence, and proteins involved with flower development. All of these data increase our understanding of the role of miRNAs in the complex regulation of drought stress in field-grown sugarcane, providing valuable tools to develop new sugarcane cultivars tolerant to drought stress.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-012-1795-7) contains supplementary material, which is available to authorized users.

Prediction of Conserved Precursors of miRNAs and Their Mature Forms by Integrating Position-Specific Structural Features

MicroRNA (miRNA) precursor hairpins have a unique secondary structure, nucleotide length, and nucleotide content that are in most cases evolutionarily conserved. The aim of this study was to utilize position-specific features of miRNA hairpins to improve their identification. To this end, we defined the evolutionary and structurally conserved features in each position of miRNA hairpins with heuristically derived values, which were successfully integrated using a probabilistic framework. Our method, miRRim2, can not only accurately detect miRNA hairpins, but infer the location of a mature miRNA sequence. To evaluate the accuracy of miRRim2, we designed a cross validation test in which the whole human genome was used for evaluation. miRRim2 could more accurately detect miRNA hairpins than the other computational predictions that had been performed on the human genome, and detect the position of the 5′-end of mature miRNAs with sensitivity and positive predictive value (PPV) above 0.4. To further evaluate miRRim2 on independent data, we applied it to the Ciona intestinalis genome. Our method detected 47 known miRNA hairpins among top 115 candidates, and pinpointed the 5′-end of mature miRNAs with sensitivity and PPV about 0.4. When our results were compared with deep-sequencing reads of small RNA libraries from Ciona intestinalis cells, we found several candidates in which the predicted mature miRNAs were in good accordance with deep-sequencing results.

Identification of conserved and novel microRNAs in Aquilaria sinensis based on small RNA sequencing and transcriptome sequence data

Agarwood is in great demand for its high value in medicine, incense, and perfume across Asia, Middle East, and Europe. As agarwood is formed only when the Aquilaria trees are wounded or infected by some microbes, overharvesting and habitat loss are threatening some populations of agarwood-producing species. Aquilaria sinensis is such a significant economic tree species. To promote the production efficiency and protect the resource of A. sinensis, it would be critical to reveal the regulation mechanisms of stress-induced agarwood formation. MicroRNAs (miRNAs), a key gene expression regulator involved in various plant stress response and metabolic processes, might function in agarwood formation, but no report concerning miRNAs in Aquilaria is available. In this study, the small RNA high-throughput sequencing and 454 transcriptome data were adopted to identify both conserved and novel miRNAs in A. sinensis. Deep sequencing showed that the small RNA (sRNA) population of A. sinensis was complex and the length of sRNAs varied. By in silico analysis of the small RNA deep sequencing data and transcriptome data, we discovered 27 novel miRNAs in A. sinensis. Based on the mature miRNA sequence conservation, we identified 74 putative conserved miRNAs from A. sinensis and 10 of them were confirmed with hairpin forming precursor. Interestingly, a novel miRNA sequence was determined to be the miRNA* of asi-miR408, but with accumulation much higher than asi-miR408. The expression levels of ten stress-responsive miRNAs were examined during the time-course after wound treatment. Eight were shown to be wound-responsive. This not only shows the existence of miRNAs in this Asian economically significant tree species but also indicated its critical role in stress-induced agarwood formation. The highly accumulated miRNA* of asi-miR408 implied miRNA*s would be functional as well as miRNAs in plants.

Dynamic MicroRNA Gene Transcription and Processing during T Cell Development

By disrupting microRNA (miRNA) biogenesis, we previously showed that this pathway is critical for the differentiation and function of T cells. Although various cloning studies have shown that many miRNAs are expressed during T cell development, and in a dynamic manner, it was unclear how comprehensive these earlier analyses were. We therefore decided to profile miRNA expression by next generation sequencing. Furthermore, we profiled miRNA expression starting from the hematopoietic stem cell. This analysis revealed that miRNA expression during T cell development is extremely dynamic, with 645 miRNAs sequenced, and the expression of some varying by as much as 3 orders of magnitude. Furthermore, changes in precursor processing led to altered mature miRNA sequences. We also analyzed the structures of the primary miRNA transcripts expressed in T cells and found that many were extremely long. The longest was pri-mir-29b-1/29a at ∼168 kb. All the long pri-miRNAs also displayed extensive splicing. Our findings indicate that miRNA expression during T cell development is both a highly dynamic and a highly regulated process.

114 A Highly Sensitive and Specific Universal Mirna Profiling Method

BackgroundmiRNAs can be used as robust biomarkers for diagnosis, staging, prognosis and the response to therapy in various diseases. Although a wide spectrum of miRNA detection techniques have been developed, none can accurately and sensitively perform genome-wide high-throughput miRNA profiling (Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, et al 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33:e179). This problem stems from that miRNAs are only ∼22 bases, and multiple species of nucleic acids that contain the mature miRNA sequences are present in the total RNA samples that are usually used for miRNA detection.MethodsA novel RT-qPCR miRNA assay (UQmiR, universally quantitating miRNA) was developed to overcome the difficulty. This assay requires only one RT reaction and one universal set of multiple hydrolysis probes to detect all miRNAs, using one universal RT primer, a common reverse primer, and individual miRNA-specific forward primers. A computer program (MSPPD, miRNA-specific primer and probe designer) was developed for the assay.ResultsThe UQmiR has the advantages, but not the disadvantages, of the 2 mostly used miRNA assays. It has the specificity of hydrolysis probe assay and the universal detection of SYBR Green assay. This assay is more sensitive and specific than the commercially available hydrolysis probe assay and SYBR Green assay. Using this method, we have successfully detected 91 out of 96 miRNAs in 0.8 mL of plasma for each miRNA.ConclusionsThis approach affords a highly specific, sensitive, economical and convenient system to profile the expression of all known miRNAs.

Role of variations within microRNA-binding sites in cancer

Over 2000 microRNA (miRNA) sequences from different species have been submitted to the miRBase, the central online repository for miRNAs, making a total of 5071 miRNA loci, expressing 5922 distinct mature miRNA sequences. In this review, we have addressed the importance of the genetic variations in humans affecting miRNAs, their target genes and the genes involved in miRNA processing for individual risk of cancer, with particular emphasis on colorectal cancer. In fact, the number of studies suggesting that individual predisposition to cancer is modulated by genetic polymorphisms affecting the biogenesis of miRNA and the interaction between miRNAs and targets has risen steeply in the last few years. We also report the first evidence that variant alleles of single-nucleotide polymorphisms (SNPs) within miRNA genes and miRNA targets, previously associated with the risk of cancer, behave differently when tested in functional studies. The SNPs belonging to the miRNA world are certainly contributing to new insights in the field of the genetic predisposition to disease.

Computational identification of microRNA gene loci and precursor microRNA sequences in CHO cell lines.

MicroRNAs (miRNAs) have recently entered Chinese hamster ovary (CHO) cell culture technology, due to their severe impact on the regulation of cellular phenotypes. Applications of miRNAs that are envisioned range from biomarkers of favorable phenotypes to cell engineering targets. These applications, however, require a profound knowledge of miRNA sequences and their genomic organization, which exceeds the currently available information of ~400 conserved mature CHO miRNA sequences. Based on these recently published sequences and two independent CHO-K1 genome assemblies, this publication describes the computational identification of CHO miRNA genomic loci. Using BLAST alignment, 415 previously reported CHO miRNAs were mapped to the reference genomes, and subsequently assigned to a distinct genomic miRNA locus. Sequences of the respective precursor-miRNAs were extracted from both reference genomes, folded in silico to verify correct structures and cross-compared. In the end, 212 genomic loci and pre-miRNA sequences representing 319 expressed mature miRNAs (approximately 50% of miRNAs represented matching pairs of 5' and 3' miRNAs) were submitted to the miRBase miRNA repository. As a proof-of-principle for the usability of the published genomic loci, four likely polycistronic miRNA cluster were chosen for PCR amplification using CHO-K1 and DHFR (-) genomic DNA. Overall, these data on the genomic context of miRNA expression in CHO will simplify the development of tools employing stable overexpression or deletion of miRNAs, allow the identification of miRNA promoters and improve detection methods such as microarrays.

Genetic polymorphisms and microRNAs: new direction in molecular epidemiology of solid cancer

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression. Single nucleotide polymorphisms (SNPs) may occur in miRNA biogenesis pathway genes, primary miRNA, pre-miRNA or a mature miRNA sequence. Such polymorphisms may be functional with respect to biogenesis and actions of mature miRNA. Specific SNPs were identified in predicted miRNA target sites within 3′ untranslated regions of mRNAs. These SNPs have a potential to affect the efficiency of miRNA binding to the target sites or can create or disrupt binding sites. Resulting gene dysregulation may involve changes in phenotype and may eventually prove critical for the susceptibility to cancer and its onset as well as for estimates of prognosis and therapy response. In this review, we provide a comprehensive list of potentially functional miRNA-related SNPs and summarize their importance as candidate cancer biomarkers.

High Mir-221 Expression Is Associated with Poorer Treatment Outcome of Patients with T-Cell Acute Lymphoblastic Leukemia

Abstract 1439T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cells that accounts about 15% of pediatric and 25% of adult ALL cases. In the last years, several clinical and laboratory features have been described as prognostic markers; nevertheless, with intensification of therapy most of them have lost their predictive value. MicroRNA (miRNA) expression analysis has proved to be an useful tool for identifying specific subsets of cancer patients with relevant cytogenetic, laboratorial and clinical features. The aim of the present study was to determine if miRNAs may be useful markers in T-ALL. First, we performed a supervised analysis comparing the miRNA expression profile of T-ALL blasts from 36 T-ALL/CD56− and 12 T-ALL/CD56+. We selected CD56 as prognostic marker based on our previous report showing that the disease-free survival (DFS) of T-ALL/CD56+ patients was of 28.5 months compared to 69.8 in the CD56− group. Also patients tended to be older and to present normal platelet counts in the T-LLA/CD56+ group. We used the Taqman MicroRNA Assay Human Panel (Applied Biosystems) to perform a screening of 164 knowledge mature miRNA sequences using specific primers and probes according to manufacturer instructions. Total RNA input was normalized based on the geometric means of Ct values obtained from four endogenous RNAs. All reactions were run in duplicate and a coefficient of variation greater than 5% was used as an exclusion factor (seven miRNAs were excluded). The fold change was calculated using comparative 2−δCt method. We have identified a set of 14 miRNAs differentially expressed, of which miR-374 and miR-221 best distinguished T-ALL/CD56+ from T-ALL/CD56− blasts. Based on this profile, we selected miR-221 and miR-374 as potential markers and quantified their expression in the same samples using RQ-PCR. Patients were stratified as high and low expression using the median value as cut off. We detected a significant association between the miR-221 high expression and poorer treatment outcome. On the contrary, miR-374 expression levels were not associated with treatment outcome. We evaluate the impact of age, white blood cell counts, CD56 and miR221 expression on overall survival (OS). Age and miR-221 were the only ones found to be significant. The estimate 5-year OS (mean and confidence interval 95%) was of 67.0 ± 10.3% in the group of patients expressing miR-221 below the cut-off value, whereas this value was of 28.5 ± 14.5% in the alternative group. Even among T-ALL/CD56− patients, the higher expression of miR-221 was significantly associated with poorer outcome. Our data suggest that miR-221 play an important role in T-ALL and its regulation may represent a potential therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Microarray Analysis of Differentially Expressed microRNAs in Allergic Rhinitis

Allergic rhinitis (AR) is a common disease characterized by chronic inflammation of the nasal mucosa, but we have not fully understood the mechanism responsible for the development of AR. MicroRNAs (miRNAs) are short endogenous noncoding RNAs regulating protein translation through a mechanism known as RNA interference. To understand the molecular mechanisms of miRNA involved in the pathogenesis of AR, expressed miRNAs in AR were investigated through genomewide microarray analysis. Mammalian miRNA microarrays containing whole human mature and precursor miRNA sequences were used for analyzing eight samples of nasal mucosa of AR and eight samples of nonallergic patients. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of some different expressed miRNAs was used to confirm the array results. The miRNA microarray chip analysis identified 421 miRNAs differentially expressed in the nasal mucosa of AR, and a total of 9 miRNAs were identified in the AR group with twofold change compared with control samples (p < 0.05). These included up-regulated miRNAs, hsa-hsa-miR-7, and hsa-miRPlus-E1194, and down-regulated miRNAs, hsa-miR-498, hsa-miR-187, hsa-miR-874, hsa-miR-143, hsa-miR-886-3p, hsa-miR-224, and hsa-miR-767-5p. RT-PCR results also confirmed that part of differentially expressed miRNAs as hsa-miR-224, hsa-miR-187, and hsa-miR-143 were down-regulated in AR. The report indicated that many miRNA expressions were altered in AR and differentially expressed miRNAs appear to be involved in the development of AR. The study of miRNAs may lead to a better understanding about the roles of identified miRNAs in the pathogenesis of AR; this would be considered in future therapeutic strategies.

A MicroRNA Catalog of Swine Umbilical Vein Endothelial Cells Identified by Deep Sequencing

MicroRNAs (miRNAs) are endogenous ∼22 nt RNAs that play important regulatory roles in targeting mRNAs for cleavage or translational repression. Despite the discovery of increasing numbers of human and mouse miRNAs, little is known about miRNAs from pig. In this study, we sought to extend the repertoire of porcine small regulatory RNAs using Solexa sequencing. We sequenced a library of small RNAs prepared from immortalized swine umbilical vein endothelial cells (SUVECs). We produced over 13.6 million short sequence reads, of which 8547658 perfectly mapped to the pig genome. A bioinformatics pipeline was used to identify authentic mature miRNA sequences. We identified 154 porcine miRNA genes, among which 146 were conserved across species, and 8 were pig-specific miRNA genes. The 146 miRNA genes encoded 116 conserved mature miRNAs and 66 miRNA*. The 8 pig-specific miRNA genes encoded 4 mature miRNAs. Four potential novel miRNAs were identified. The secondary structures of the 154 miRNA genes were predicted; 13 miRNAs have 2 structures, and miR-9 and miR-199 have 4 and 3 structures, respectively. 36 miRNAs were organized into 19 compact clusters. miR-206, miR-21 and miR-378 were the relatively highly expressed miRNAs. In conclusion, Solexa sequencing allowed the successful discovery of known and novel porcine miRNAs with high accuracy and efficiency. Furthermore, our results supply new data to the somewhat insufficient pig miRBase, and are useful for investigating features of the blood-brain barrier, vascular diseases and inflammation.

Uncovering New Functions for MicroRNAs in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, microRNA (miRNA) regulation of development was first observed in the striking abnormalities of lin-4 and let-7 loss of function mutants. However, after these first two miRNA mutant phenotypes were described, progress on the identification of miRNA functions in worms slowed considerably. Recent advances reveal new functions for miRNAs in embryonic and larval development as well as in the regulation of lifespan and stress response. Results from a combination ofcomputational, biochemical, and genetic approaches have deepened our understanding of miRNA regulation of target mRNAs and support the hypothesis that miRNAs have an important role in ensuring the robustness of developmental and physiological pathways.

Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression

MicroRNA (miRNA) plays essential roles in regulating gene expression, but miRNA delivery remains a hurdle, thus entailing a vector system for efficient transfer. Baculovirus emerges as a promising gene delivery vector but its inherent transient expression restricts its applications in some scenarios. Therefore, this study primarily aimed to develop baculovirus as a miRNA expression vector for prolonged gene suppression. We constructed recombinant baculoviruses carrying artificial egfp-targeting miRNA sequences within the miR155 backbone, which after expression by the cytomegalovirus promoter could knockdown the enhanced green fluorescent protein (EGFP) expression in a sequence- and dose-dependent manner. By swapping the mature miRNA sequences, the baculovirus miRNA shuttle effectively repressed the overexpression of endogenous TNF-α in arthritic synoviocytes without inducing apoptosis. To prolong the baculovirus-mediated expression, we further developed a hybrid baculovirus vector that exploited the Sleeping Beauty (SB) transposon for gene integration and sustained miRNA expression. The hybrid baculovirus vector that combined the miR155 scaffold and SB transposon effectively repressed the transgene expression for a prolonged period of time, hence diversifying the applications of baculovirus to indications necessitating prolonged gene regulation such as arthritis.