Orthologous microRNA genes are located in cancer-associated genomic regions in human and mouse

Background

MicroRNAs (miRNAs) are short non-coding RNAs that regulate differentiation and development in many organisms and play an important role in cancer.

Methodology/Principal Findings

Using a public database of mapped retroviral insertion sites from various mouse models of cancer we demonstrate that MLV-derived retroviral inserts are enriched in close proximity to mouse miRNA loci. Clustered inserts from cancer-associated regions (Common Integration Sites, CIS) have a higher association with miRNAs than non-clustered inserts. Ten CIS-associated miRNA loci containing 22 miRNAs are located within 10 kb of known CIS insertions. Only one CIS-associated miRNA locus overlaps a RefSeq protein-coding gene and six loci are located more than 10 kb from any RefSeq gene. CIS-associated miRNAs on average are more conserved in vertebrates than miRNAs associated with non-CIS inserts and their human homologs are also located in regions perturbed in cancer. In addition we show that miRNA genes are enriched around promoter and/or terminator regions of RefSeq genes in both mouse and human.

Conclusions/Significance

We provide a list of ten miRNA loci potentially involved in the development of blood cancer or brain tumors. There is independent experimental support from other studies for the involvement of miRNAs from at least three CIS-associated miRNA loci in cancer development.     

Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby (Macropus eugenii)

Background

Lactation is a key aspect of mammalian evolution for adaptation of various reproductive strategies along different mammalian lineages. Marsupials, such as tammar wallaby, adopted a short gestation and a relatively long lactation cycle, the newborn is immature at birth and significant development occurs postnatally during lactation. Continuous changes of tammar milk composition may contribute to development and immune protection of pouch young. Here, in order to address the putative contribution of newly identified secretory milk miRNA in these processes, high throughput sequencing of miRNAs collected from tammar milk at different time points of lactation was conducted. A comparative analysis was performed to find distribution of miRNA in milk and blood serum of lactating wallaby.

Results

Results showed that high levels of miRNA secreted in milk and allowed the identification of differentially expressed milk miRNAs during the lactation cycle as putative markers of mammary gland activity and functional candidate signals to assist growth and timed development of the young. Comparative analysis of miRNA distribution in milk and blood serum suggests that milk miRNAs are primarily expressed from mammary gland rather than transferred from maternal circulating blood, likely through a new putative exosomal secretory pathway. In contrast, highly expressed milk miRNAs could be detected at significantly higher levels in neonate blood serum in comparison to adult blood, suggesting milk miRNAs may be absorbed through the gut of the young.

Conclusion

The function of miRNA in mammary gland development and secretory activity has been proposed, but results from the current study also support a differential role of milk miRNA in regulation of development in the pouch young, revealing a new potential molecular communication between mother and young during mammalian lactation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1012) contains supplementary material, which is available to authorized users.     

Microarray-Based Approach Identifies Differentially Expressed MicroRNAs in Porcine Sexually Immature and Mature Testes

Background

MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood.

Methodology

We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR.

Conclusions

Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis.     

Deep Sequencing and Microarray Hybridization Identify Conserved and Species-Specific MicroRNAs during Somatic Embryogenesis in Hybrid Yellow Poplar

Background

To date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense).

Methodology/Principal Findings

A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology.

Conclusions

Solexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.     

Assessing the utility of thermodynamic features for microRNA target prediction under relaxed seed and no conservation requirements

Background

Many computational microRNA target prediction tools are focused on several key features, including complementarity to 5′seed of miRNAs and evolutionary conservation. While these features allow for successful target identification, not all miRNA target sites are conserved and adhere to canonical seed complementarity. Several studies have propagated the use of energy features of mRNA:miRNA duplexes as an alternative feature. However, different independent evaluations reported conflicting results on the reliability of energy-based predictions. Here, we reassess the usefulness of energy features for mammalian target prediction, aiming to relax or eliminate the need for perfect seed matches and conservation requirement.

Methodology/Principal Findings

We detect significant differences of energy features at experimentally supported human miRNA target sites and at genome-wide sites of AGO protein interaction. This trend is confirmed on datasets that assay the effect of miRNAs on mRNA and protein expression changes, and a simple linear regression model leads to significant correlation of predicted versus observed expression change. Compared to 6-mer seed matches as baseline, application of our energy-based model leads to ∼3–5-fold enrichment on highly down-regulated targets, and allows for prediction of strictly imperfect targets with enrichment above baseline.

Conclusions/Significance

In conclusion, our results indicate significant promise for energy-based miRNA target prediction that includes a broader range of targets without having to use conservation or impose stringent seed match rules.     

Mammalian conserved ADAR targets comprise only a small fragment of the human editosome

Background

ADAR proteins are among the most extensively studied RNA binding proteins. They bind to their target and deaminate specific adenosines to inosines. ADAR activity is essential, and the editing of a subset of their targets is critical for viability. Recently, a huge number of novel ADAR targets were detected by analyzing next generation sequencing data. Most of these novel editing sites are located in lineage-specific genomic repeats, probably a result of overactivity of editing enzymes, thus masking the functional sites. In this study we aim to identify the set of mammalian conserved ADAR targets.

Results

We used RNA sequencing data from human, mouse, rat, cow, opossum, and platypus to define the conserved mammalian set of ADAR targets. We found that the conserved mammalian editing sites are surprisingly small in number and have unique characteristics that distinguish them from non-conserved ones. The sites that constitute the set have a distinct genomic distribution, tend to be located in genes encoding neurotransmitter receptors or other synapse related proteins, and have higher editing and expression levels. We also found a high consistency of editing levels of this set within mice strains and between human and mouse. Tight regulation of editing in these sites across strains and species implies their functional importance.

Conclusions

Despite the discovery of numerous editing targets, only a small number of them are conserved within mammalian evolution. These sites are extremely highly conserved and exhibit unique features, such as tight regulation, and probably play a pivotal role in mammalian biology.     

Mirna expression profiles identify drivers in colorectal and pancreatic cancers

Background and Aim

Altered expression of microRNAs (miRNAs) hallmarks many cancer types. The study of the associations of miRNA expression profile and cancer phenotype could help identify the links between deregulation of miRNA expression and oncogenic pathways.

Methods

Expression profiling of 866 human miRNAs in 19 colorectal and 17 pancreatic cancers and in matched adjacent normal tissues was investigated. Classical paired t-test and random forest analyses were applied to identify miRNAs associated with tissue-specific tumors. Network analysis based on a computational approach to mine associations between cancer types and miRNAs was performed.

Results

The merge between the two statistical methods used to intersect the miRNAs differentially expressed in colon and pancreatic cancers allowed the identification of cancer-specific miRNA alterations. By miRNA-network analysis, tissue-specific patterns of miRNA deregulation were traced: the driving miRNAs were miR-195, miR-1280, miR-140-3p and miR-1246 in colorectal tumors, and miR-103, miR-23a and miR-15b in pancreatic cancers.

Conclusion

MiRNA expression profiles may identify cancer-specific signatures and potentially useful biomarkers for the diagnosis of tissue specific cancers. miRNA-network analysis help identify altered miRNA regulatory networks that could play a role in tumor pathogenesis.     

MiRNAs Which Target CD3 Subunits Could Be Potential Biomarkers for Cancers

Background

T-cells play an important role in the immune response and are activated in response to the presentation of antigens bound to major histocompatibility complex (MHC) molecules participating with the T-cell receptor (TCR). T-cell receptor complexes also contain four CD3 (cluster of differentiation 3) subunits. The TCR-CD3 complex is vital for T-cell development and plays an important role in intervening cell recognition events. Since microRNAs (miRNAs) are highly stable in blood serum, some of which may target CD3 molecules, they could serve as good biomarkers for early cancer detection. The aim of this study was to see whether there is a relationship between cancers and the amount of miRNAs -targeted CD3 molecules.

Methods

Bioinformatics tools were used in order to predict the miRNA targets for these genes. Subsequently, these highly conserved miRNAs were evaluated to see if they are implicated in various kinds of cancers. Consequently, human disease databases were used. According to the latest research, this study attempted to investigate the possible down- or upregulation of miRNAs cancer patients.

Results

We identified miRNAs which target genes producing CD3 subunit molecules. The most conserved miRNAs were identified for the CD3G gene, while CD247 and CD3EAP genes had the least number and there were no conserved miRNA associated with the CD3D gene. Some of these miRNAs were found to be responsible for different cancers, following a certain pattern.

Conclusions

It is highly likely that miRNAs affect the CD3 molecules, impairing the immune system, recognizing and destroying cancer tumor; hence, they can be used as suitable biomarkers in distinguishing cancer in the very early stages of its development.     

Identification and Characterization of Novel MicroRNAs from Schistosoma japonicum

Background

Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs.

Methodology and Results

We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host.

Conclusions

Authentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.     

The Fate of miRNA* Strand through Evolutionary Analysis: Implication for Degradation As Merely Carrier Strand or Potential Regulatory Molecule?

Background

During typical microRNA (miRNA) biogenesis, one strand of a ∼22 nt RNA duplex is preferentially selected for entry into a silencing complex, whereas the other strand, known as the passenger strand or miRNA* strand, is degraded. Recently, some miRNA* sequences were reported as guide miRNAs with abundant expression. Here, we intended to discover evolutionary implication of the fate of miRNA* strand by analyzing miRNA/miRNA* sequences across vertebrates.

Principal Findings

Mature miRNAs based on gene families were well conserved especially for their seed sequences across vertebrates, while their passenger strands always showed various divergence patterns. The divergence mainly resulted from divergence of different animal species, homologous miRNA genes and multicopy miRNA hairpin precursors. Some miRNA* sequences were phylogenetically conserved in seed and anchor sequences similar to mature miRNAs, while others revealed high levels of nucleotide divergence despite some of their partners were highly conserved. Most of those miRNA precursors that could generate abundant miRNAs from both strands always were well conserved in sequences of miR-#-5p and miR-#-3p, especially for their seed sequences.

Conclusions

The final fate of miRNA* strand, either degraded as merely carrier strand or expressed abundantly as potential functional guide miRNA, may be destined across evolution. Well-conserved miRNA* strands, particularly conservation in seed sequences, maybe afford potential opportunities for contributing to regulation network. The study will broaden our understanding of potential functional miRNA* species.     

Diversity,expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants

Background

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved.

Results

We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors.

Conclusions

Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show that rapid evolution and likely loss of expression of miR168 isoforms in tobacco is related to the insertion of MITE-like transposons between miRNA and miRNA* sequences, a possible mechanism showing how miRNAs are lost in few plant lineages even though other close relatives have abundantly expressing miRNAs.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1049) contains supplementary material, which is available to authorized users.     

Alterations in Circulating miRNA Levels following Early-Stage Estrogen Receptor-Positive Breast Cancer Resection in Post-Menopausal Women

Introduction

Circulating microRNAs (miRNAs) exhibit remarkable stability and may serve as biomarkers in several clinical cancer settings. The aim of this study was to investigate changes in the levels of specific circulating miRNA following breast cancer surgery and evaluate whether these alterations were also observed in an independent data set.

Methods

Global miRNA analysis was performed on prospectively collected serum samples from 24 post-menopausal women with estrogen receptor-positive early-stage breast cancer before surgery and 3 weeks after tumor resection using global LNA-based quantitative real-time PCR (qPCR).

Results

Numbers of specific miRNAs detected in the samples ranged from 142 to 161, with 107 miRNAs detectable in all samples. After correction for multiple comparisons, 3 circulating miRNAs (miR-338-3p, miR-223 and miR-148a) exhibited significantly lower, and 1 miRNA (miR-107) higher levels in post-operative vs. pre-operative samples (p<0.05). No miRNAs were consistently undetectable in the post-operative samples compared to the pre-operative samples. Subsequently, our findings were compared to a dataset from a comparable patient population analyzed using similar study design and the same qPCR profiling platform, resulting in limited agreement.

Conclusions

A panel of 4 circulating miRNAs exhibited significantly altered levels following radical resection of primary ER+ breast cancers in post-menopausal women. These specific miRNAs may be involved in tumorigenesis and could potentially be used to monitor whether all cancer cells have been removed at surgery and/or, subsequently, whether the patients develop recurrence.     

Identification of miRNAs in a Liver of a Human Fetus by a Modified Method

Background

miRNAs are 17–25 nucleotides long RNA molecules that have been found to regulate gene expression in human cells. There are studies showing that different groups of miRNAs are involved in development of different tissues. In hepatocytes there are reported particular types of miRNAs that regulate gene expression.

Methods

We established a human fetal liver cDNA library by a modified cloning protocol. Then plasmid isolation from the colonies was performed. After sequencing and database searching, the miRNAs were recognized. RT-PCR and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA.

Results

One novel miRNA was discovered, together with another 35 previously-known miRNAs in the fetal liver. Some of them existed in variants. The miRNAs identified were validated by RT-PCR and sequencing. Quantitative analysis showed that they have variable expression.

Conclusion

Our results indicate that a special group of miRNAs may play an important role in fetal liver development in a synergistic manner.     

Comprehensive miRNA Expression Analysis in Peripheral Blood Can Diagnose Liver Disease

Background

miRNAs circulating in the blood in a cell-free form have been acknowledged for their potential as readily accessible disease markers. Presently, histological examination is the golden standard for diagnosing and grading liver disease, therefore non-invasive options are desirable. Here, we investigated if miRNA expression profile in exosome rich fractionated serum could be useful for determining the disease parameters in patients with chronic hepatitis C (CHC).

Methodology

Exosome rich fractionated RNA was extracted from the serum of 64 CHC and 24 controls with normal liver (NL). Extracted RNA was subjected to miRNA profiling by microarray and real-time qPCR analysis. The miRNA expression profiles from 4 chronic hepatitis B (CHB) and 12 non alcoholic steatohepatitis (NASH) patients were also established. The resulting miRNA expression was compared to the stage or grade of CHC determined by blood examination and histological inspection.

Principal Findings

miRNAs implicated in chronic liver disease and inflammation showed expression profiles that differed from those in NL and varied among the types and grades of liver diseases. Using the expression patterns of nine miRNAs, we classified CHC and NL with 96.59% accuracy. Additionally, we could link miRNA expression pattern with liver fibrosis stage and grade of liver inflammation in CHC. In particular, the miRNA expression pattern for early fibrotic stage differed greatly from that observed in high inflammation grades.

Conclusions

We demonstrated that miRNA expression pattern in exosome rich fractionated serum shows a high potential as a biomarker for diagnosing the grade and stage of liver diseases.