Computational analysis of mRNA expression profiles identifies microRNA-29a/c as predictor of colorectal cancer early recurrence

Colorectal cancer (CRC) is one of the leading malignant cancers with a rapid increase in incidence and mortality. The recurrences of CRC after curative resection are sometimes unavoidable and often take place within the first year after surgery. MicroRNAs may serve as biomarkers to predict early recurrence of CRC, but identifying them from over 1,400 known human microRNAs is challenging and costly. An alternative approach is to analyze existing expression data of messenger RNAs (mRNAs) because generally speaking the expression levels of microRNAs and their target mRNAs are inversely correlated. In this study, we extracted six mRNA expression data of CRC in four studies (GSE12032, GSE17538, GSE4526 and GSE17181) from the gene expression omnibus (GEO). We inferred microRNA expression profiles and performed computational analysis to identify microRNAs associated with CRC recurrence using the IMRE method based on the MicroCosm database that includes 568,071 microRNA-target connections between 711 microRNAs and 20,884 gene targets. Two microRNAs, miR-29a and miR-29c, were disclosed and further meta-analysis of the six mRNA expression datasets showed that these two microRNAs were highly significant based on the Fisher p-value combination (p = 9.14 × 10(-9) for miR-29a and p = 1.14 × 10(-6) for miR-29c). Furthermore, these two microRNAs were experimentally tested in 78 human CRC samples to validate their effect on early recurrence. Our empirical results showed that the two microRNAs were significantly down-regulated (p = 0.007 for miR-29a and p = 0.007 for miR-29c) in the early-recurrence patients. This study shows the feasibility of using mRNA profiles to indicate microRNAs. We also shows miR-29a/c could be potential biomarkers for CRC early recurrence.     

Misexpression of Testicular MicroRNA in Sterile Xenopus Hybrids Points to Tetrapod-Specific MicroRNAs Associated with Male Fertility

Spermatogenesis is one of the most complex biological processes undergone by any organism, making it susceptible to perturbations that result in male sterility. Research has demonstrated that mutant phenotypes can be obtained from the disruption of epigenetic modifications, which are commonly microRNA guided. Employing the Xenopus system, whereby homogametic interspecies males are always sterile, thus violating Haldane's Rule, we deep-sequenced testes-specific small-RNAs to identify microRNAs most frequently misexpressed between sterile hybrids and their fertile parental taxa. Using these data, we cross-referenced our expression information with previously published mouse (Mus musculus) data and identified a subset of seven microRNAs common to both (miR-338, miR-222, miR-18, miR-30, miR-10, miR-196, and miR-365). We propose that these microRNAs are likely critical for spermatogenesis in all tetrapods, having retained testicular expression across ~350 million years of evolution (Amphibian-Mammal split). Gene targets of six of these microRNAs are known, and all the six associate with zinc and zinc finger proteins (both previously found critical in male fertility), and three with Hox genes (some of which have also previously been deemed critical for testicular development and male fertility). Expression information for these targets revealed that all those associated with zinc have previously been found to express in mammalian testes. One Hox target has known mammalian testicular expression, two have close relatives with known mammalian testicular expression, and two more are associated with proteins known to have mammalian testicular expression. In addition, miR-222 has prior association with spermatogenesis, and miR-30 has been found to be abundantly expressed in both mouse and human testes.     

Allosteric regulation of noncoding RNA function by microRNAs

HSUR1 and HSUR2, two noncoding RNAs expressed by the oncogenic Herpesvirus saimiri, bind host microRNAs miR-142-3p, miR-16, and miR-27 with different purposes. While binding of miR-27 to HSUR1 triggers the degradation of the microRNA, miR-16 is tethered by HSUR2 to target host mRNAs to repress their expression. Here we show that the interaction with miR-142-3p is required for the activity of both HSURs. Coimmunoprecipitation experiments revealed that miR-142-3p allosterically regulates the binding of miR-27 and miR-16 to HSUR1 and HSUR2, respectively. The binding of two different miRNAs to each HSUR is not cooperative. HSURs can be engineered to be regulated by other miRNAs, indicating that the identity of the binding miRNA is not important for HSUR regulation. Our results uncover a mechanism for allosteric regulation of noncoding RNA function and a previously unappreciated way in which microRNAs can regulate gene expression.     

A MicroRNA Network Dysregulated in Asthma Controls IL-6 Production in Bronchial Epithelial Cells

MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-β, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-β, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-β and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying disease.     

Cell Survival Following Radiation Exposure Requires miR-525-3p Mediated Suppression of ARRB1 and TXN1

Background

microRNAs (miRNAs) are non-coding RNAs that alter the stability and translation efficiency of messenger RNAs. Ionizing radiation (IR) induces rapid and selective changes in miRNA expression. Depletion of the miRNA processing enzymes Dicer or Ago2 reduces the capacity of cells to survive radiation exposure. Elucidation of critical radiation-regulated miRNAs and their target proteins offers a promising approach to identify new targets to increase the therapeutic effectiveness of the radiation treatment of cancer.

Principal Findings

Expression of miR-525-3p is rapidly up-regulated in response to radiation. Manipulation of miR-525-3p expression in irradiated cells confirmed that this miRNA mediates the radiosensitivity of a variety of non-transformed (RPE, HUVEC) and tumor-derived cell lines (HeLa, U2-Os, EA.hy926) cell lines. Thus, anti-miR-525-3p mediated inhibition of the increase in miR-525-3p elevated radiosensitivity, while overexpression of precursor miR-525-3p conferred radioresistance. Using a proteomic approach we identified 21 radiation-regulated proteins, of which 14 were found to be candidate targets for miR-525-3p-mediated repression. Luciferase reporter assays confirmed that nine of these were indeed direct targets of miR-525-3p repression. Individual analysis of these direct targets by RNAi-mediated knockdown established that ARRB1, TXN1 and HSPA9 are essential miR-525-3p-dependent regulators of radiation sensitivity.

Conclusion

The transient up-regulation of miR-525-3p, and the resultant repression of its direct targets ARRB1, TXN1 and HSPA9, is required for cell survival following irradiation. The conserved function of miR-525-3p across several cell types makes this microRNA pathway a promising target for modifying the efficacy of radiotherapy.     

Evolution of coding and non-coding genes in HOX clusters of a marsupial

ABSTRACT: BACKGROUND: The HOX gene clusters are thought to be highly conserved amongst mammals and othervertebrates, but the long non-coding RNAs have only been studied in detail in human andmouse. The sequencing of the kangaroo genome provides an opportunity to use comparativeanalyses to compare the HOX clusters of a mammal with a distinct body plan to those ofother mammals. RESULTS: Here we report a comparative analysis of HOX gene clusters between an Australian marsupialof the kangaroo family and the eutherians. There was a strikingly high level of conservationof HOX gene sequence and structure and non-protein coding genes including the microRNAsmiRNA-196a, miRNA-196b, miRNA-10a and miRNA-10b and the long non-coding RNAsHOTAIR, HOTAIRM1 and HOXA11AS that play critical roles in regulating gene expressionand controlling development. By microRNA deep sequencing and comparative genomicanalyses, two conserved microRNAs (miR-10a and miR-10b) were identified and one newcandidate microRNA with typical hairpin precursor structure that is expressed in bothfibroblasts and testes was found. The prediction of microRNA target analysis showed thatseveral known microRNA targets, such as mir-10, mir-414 and mir-464, were found in thetammar HOX clusters. In addition, several novel and putative miRNAs were identified thatoriginated from elsewhere in the tammar genome and that target the tammar HOXB andHOXD clusters. CONCLUSIONS: This study confirms that the emergence of known long non-coding RNAs in the HOXclusters clearly predate the marsupial-eutherian divergence 160 Ma ago. It also identified anew potentially functional microRNA as well as conserved miRNAs. These non-codingRNAs may participate in the regulation of HOX genes to influence the body plan of thismarsupial.     

Identification of let-7a-2-3p or/and miR-188-5p as Prognostic Biomarkers in Cytogenetically Normal Acute Myeloid Leukemia

Cytogenetically normal acute myeloid leukemia (CN-AML) is the largest and most heterogeneous AML subgroup. It lacks sensitive and specific biomarkers. Emerging evidences have suggested that microRNAs are involved in the pathogenesis of various leukemias. This paper evaluated the association between microRNA expression and prognostic outcome for CN-AML, based on the RNA/microRNA sequencing data of CN-AML patients. High let-7a-2-3p expression and low miR-188-5p expression were identified to be significantly associated with longer overall survival (OS) and event free survival (EFS) for CN-AML, independently or in a combined way. Their prognostic values were further confirmed in European Leukemia Net (ELN) genetic categories. Also, in multivariable analysis with other known risk factors, high let-7a-2-3p and low miR-188-5p expression remained to be associated with longer OS and EFS. In addition, the prognostic value of these two microRNAs was confirmed in patients who received hematopoietic stem cell transplantation (HSCT). To gain more biological insights of the underlying mechanisms, we derived the genome-wide differential gene/microRNA signatures associated with the expression of let-7a-2-3p and miR-188-5p. Several common microRNA signatures indicating favorable outcome in previous studies were up-regulated in both high let-7a-2-3p expressers and low miR-188-5p expressers, including miR-135a, miR-335 and miR-125b and all members of miR-181 family. Additionally, common up-regulated genes included FOSB, IGJ, SNORD50A and ZNF502, and FOSB was a known favorable signature in AML. These common signatures further confirmed the underlying common mechanisms for these two microRNAs value as favorable prognostic biomarkers. We concluded that high let-7a-2-3p and low miR-188-5p expression could be potentially used as favorably prognostic biomarkers independently or in a combined way in CN-AML patients, whether they received HSCT or not.     

MicroRNA Profiling of Pericardial Fluid Samples from Patients with Heart Failure

Aims

Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate biomarkers due to their relative stability and easy quantification from clinical samples. Pericardial fluid contains hormones secreted by the heart and is known to reflect the cardiac function. In this study, we sought to investigate whether pericardial fluid contains microRNAs and if so, whether they could be used to distinguish between different cardiovascular pathologies and disease stages.

Methods and Results

Pericardial fluid was collected from heart failure patients during open-heart surgery. MicroRNA profiles of altogether 51 patients were measured by quantitative real-time PCR (qPCR) using Exiqon human panels I and II. On the average, 256 microRNAs were detected per sample, and 70 microRNAs out of 742 profiled microRNAs were detected in every sample. The five most abundant microRNAs in pericardial fluid were miR-21-5p, miR-451a, miR-125b-5p, let-7b-5p and miR-16-5p. No specific signatures for cardiovascular pathologies or clinically assessed heart failure stages could be detected from the profiles and, overall, microRNA profiles of the samples were found to be very similar despite the heterogeneity in the study population.

Conclusion

Measured microRNA profiles did not separate the samples according to the clinical features of the patients. However, several previously identified heart failure marker microRNAs were detected. The pericardial fluid microRNA profile appeared to be a result of an active and selective secretory process indicating that microRNAs may act as paracrine signalling factors by mediating the local crosstalk between cardiac cells.     

Computational identification of microRNA targets

Recent experiments have shown that the genomes of organisms such as worm, fly, human, and mouse encode hundreds of microRNA genes. Many of these microRNAs are thought to regulate the translational expression of other genes by binding to partially complementary sites in messenger RNAs. Phenotypic and expression analysis suggests an important role of microRNAs during development. Therefore, it is of fundamental importance to identify microRNA targets. However, no experimental or computational high-throughput method for target site identification in animals has been published yet. Our main result is a new computational method that is designed to identify microRNA target sites. This method recovers with high specificity known microRNA target sites that have previously been defined experimentally. Based on these results, we present a simple model for the mechanism of microRNA target site recognition. Our model incorporates both kinetic and thermodynamic components of target recognition. When we applied our method to a set of 74 Drosophila melanogaster microRNAs, searching 3'UTR sequences of a predefined set of fly mRNAs for target sites which were evolutionary conserved between D. melanogaster and Drosophila pseudoobscura, we found that many key developmental body patterning genes such as hairy and fushi-tarazu are likely to be translationally regulated by microRNAs.     

Traumatic Brain Injury Dysregulates MicroRNAs to Modulate Cell Signaling in Rat Hippocampus

Traumatic brain injury (TBI) is a common cause for cognitive and communication problems, but the molecular and cellular mechanisms are not well understood. Epigenetic modifications, such as microRNA (miRNA) dysregulation, may underlie altered gene expression in the brain, especially hippocampus that plays a major role in spatial learning and memory and is vulnerable to TBI. To advance our understanding of miRNA in pathophysiological processes of TBI, we carried out a time-course microarray analysis of microRNA expression profile in rat ipsilateral hippocampus and examined histological changes, apoptosis and synapse ultrastructure of hippocampus post moderate TBI. We found that 10 out of 156 reliably detected miRNAs were significantly and consistently altered from one hour to seven days after injury. Bioinformatic and gene ontology analyses revealed 107 putative target genes, as well as several biological processes that might be initiated by those dysregulated miRNAs. Among those differentially expressed microRNAs, miR-144, miR-153 and miR-340-5p were confirmed to be elevated at all five time points after TBI by quantitative RT-PCR. Western blots showed three of the predicated target proteins, calcium/calmodulin-dependent serine protein kinase (CASK), nuclear factor erythroid 2-related factor 2 (NRF2) and alpha-synuclein (SNCA), were concurrently down- regulated, suggesting that miR-144, miR-153 and miR-340-5p may play important roles collaboratively in the pathogenesis of TBI-induced cognitive and memory impairments. These microRNAs might serve as potential targets for progress assessment and intervention against TBI to mitigate secondary damage to the brain.     

LncRNA TUG1 attenuates ischaemia-reperfusion-induced apoptosis of renal tubular epithelial cells by sponging miR-144-3p via targeting Nrf2

Renal ischaemia/reperfusion (I/R) injury may induce kidney damage and dysfunction, in which oxidative stress and apoptosis play important roles. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are reported to be closely related to renal I/R, but the specific molecular mechanism is still unclear. The purpose of this research was to explore the regulatory effect of lncRNA TUG1 on oxidative stress and apoptosis in renal I/R injury. This research revealed that in renal I/R injury and hypoxia/reperfusion (H/R) injury in vitro, the expression level of lncRNA TUG1 was upregulated, and oxidative stress levels and apoptosis levels were negatively correlated with the expression level of lncRNA TUG1. Using bioinformatics databases such as TargetScan and microRNA.org, microRNA-144-3p (miR-144-3p) was predicted to be involved in the association between lncRNA TUG1 and Nrf2. This study confirmed that the level of miR-144-3p was significantly reduced following renal I/R injury and H/R injury in vitro, and miR-144-3p was determined to target Nrf2 and inhibit its expression. In addition, lncRNA TUG1 can reduce the inhibitory effect of miR-144-3p on Nrf2 by sponging miR-144-3p. In summary, our research shows that lncRNA TUG1 regulates oxidative stress and apoptosis during renal I/R injury through the miR-144-3p/Nrf2 axis, which may be a new treatment target for renal I/R injury.     

Deep sequencing reveals predominant expression of miR-21 amongst the small non-coding RNAs in retinal microvascular endothelial cells

The retinal vascular endothelium is essential for angiogenesis and is involved in maintaining barrier selectivity and vascular tone. The aim of this study was to identify and quantify microRNAs and other small regulatory non-coding RNAs (ncRNAs) which may regulate these crucial functions. Primary bovine retinal microvascular endothelial cells (RMECs) provide a well-characterized in vitro system for studying angiogenesis. RNA extracted from RMECs was used to prepare a small RNA library for deep sequencing (Illumina Genome Analyzer). A total of 6.8 million reads were mapped to 250 known microRNAs in miRBase (release 16). In many cases, the most frequent isomiR differed from the sequence reported in miRBase. In addition, five novel microRNAs, 13 novel bovine orthologs of known human microRNAs and multiple new members of the miR-2284/2285 family were detected. Several ～30 nucleotide sno-miRNAs were identified, with the most highly expressed being derived from snoRNA U78. Highly expressed microRNAs previously associated with endothelial cells included miR-126 and miR-378, but the most highly expressed was miR-21, comprising more than one-third of all mapped reads. Inhibition of miR-21 with an LNA inhibitor significantly reduced proliferation, migration, and tube-forming capacity of RMECs. The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs and other small RNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in RMECs. Knockdown of miR-21 suggests a role for this microRNA in regulation of angiogenesis in the retinal microvasculature.     

MicroRNA and target protein patterns reveal physiopathological features of glioma subtypes

Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets.     

miR‐423‐5p inhibits myoblast proliferation and differentiation by targeting Sufu

Myoblast proliferation and terminal differentiation are the key steps of myogenesis. MicroRNAs are a class of small noncoding RNAs that play important roles in gene expression regulation. They negatively regulate gene expression by causing messenger RNA translational repression or target messenger RNA degradation. Here, we found that microRNA‐423‐5p (miR‐423‐5p) is highly expressed in both slow and fast muscles. Our gain‐of‐function study indicated that miR‐423‐5p actually plays a negative role in regulating myoblast proliferation and differentiation. We also found that miR‐423‐5p is able to inhibit the expression of suppressor of fused homolog to inactivate the expression of the marker genes in myoblast proliferation and differentiation. Taken together, our findings indicated miR‐423‐5p as a potential inhibitor of myogenesis by targeting suppressor of fused homolog in myoblast, and it also contributes to a better understanding of the microRNAs‐target gene regulatory network in different types of porcine muscle types and may benefit the practice of improving the meat quality in animal husbandry.