Modulated expression of human peripheral blood microRNAs from infancy to adulthood and its role in aging

Accumulating evidence suggests a role for microRNAs (miRNAs) in regulating various processes of mammalian postnatal development and aging. To investigate the changes in blood‐based miRNA expression from preterm infants to adulthood, we compared 365 miRNA expression profiles in a screening set of preterm infants and adults. Approximately one‐third of the miRNAs were constantly expressed from postnatal development to adulthood, another one‐third were differentially expressed between preterm infants and adults, and the remaining one‐third were not detectable in these two groups. Based on their expression in infants and adults, the miRNAs were categorized into five classes, and six of the seven miRNAs chosen from each class except one with age‐constant expression were confirmed in a validation set containing infants, children, and adults. Comparing the chromosomal locations of the different miRNA classes revealed two hot spots: the miRNA cluster on 14q32.31 exhibited age‐constant expression, and the one on 9q22.21 exhibited up‐regulation in adults. Furthermore, six miRNAs detectable in adults were down‐regulated in older adults, and four chosen for individual quantification were verified in the validation set. Analysis of the network functions revealed that differentially regulated miRNAs between infants and adults and miRNAs that decreased during aging shared two network functions: inflammatory disease and inflammatory response. Four expression patterns existed in the 11 miRNAs from infancy to adulthood, with a significant transition in ages 9–20 years. Our results provide an overview on the regulation pattern of blood miRNAs throughout life and the possible biological functions performed by different classes of miRNAs.     

Genome‐wide identification and characterization of microRNAs responding to ABA and GA in maize embryos during seed germination

• MicroRNAs (miRNAs) are an important class of non‐coding small RNAs that regulate the expression of target genes through mRNA cleavage or translational inhibition. Previous studies have revealed their roles in regulating seed dormancy and germination in model plants such as Arabidopsis thaliana, rice (Oryza sativa) and maize (Zea mays). However, the miRNA response to exogenous gibberellic acid (GA) and abscisic acid (ABA) during seed germination in maize has yet to be explored.
• In this study, small RNA libraries were generated and sequenced from maize embryos treated with GA, ABA or double‐distilled water as control.
• A total of 247 miRNAs (104 known and 143 novel) were identified, of which 45 known and 53 novel miRNAs were differentially expressed in embryos in the different treatment groups. In total, 74 (37 up‐regulated and 37 down‐regulated) and 55 (23 up‐regulated and 32 down‐regulated) miRNAs were expressed in response to GA and to ABA, respectively, and a total of 18 known and 38 novel miRNAs displayed differential expression between the GA‐ and ABA‐treated groups. Using bioinformatics tools, we predicted the target genes of the differentially expressed miRNAs. Using GO enrichment and KEGG pathway analysis of these targets, we showed that miRNAs differentially expressed in our samples affect genes encoding proteins involved in the peroxisome, ribosome and plant hormonal signalling pathways.
• Our results indicate that miRNA‐mediated gene expression influences the GA and ABA signalling pathways during seed germination.

     

Altered microRNA expression in bovine skeletal muscle with age

Age‐dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. We therefore performed a comprehensive investigation for miRNAs from five different periods of bovine skeletal muscle development using next‐generation small RNA sequencing. In total, 511 miRNAs, including one putatively novel miRNA, were identified. Thirty‐six miRNAs were differentially expressed between prenatal and postnatal stages of muscle development including several myomiRs (miR‐1, miR‐206 and let‐7 families). Compared with miRNA expression between different muscle tissues, 14 miRNAs were up‐regulated and 22 miRNAs were down‐regulated in the muscle of postnatal stage. In addition, a novel miRNA was predicted and submitted to the miRBase database as bta‐mir‐10020. A dual luciferase reporter assay was used to demonstrate that bta‐mir‐10020 directly targeted the 3′‐UTR of the bovine ANGPT1 gene. The overexpression of bta‐mir‐10020 significantly decreased the DsRed fluorescence in the wild‐type expression cassette compared to the mutant type. Using three computational approaches – miranda , pita and rnahybrid – these differentially expressed miRNAs were also predicted to target 3609 bovine genes. Disease and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Our miRNA expression analysis findings from different states of muscle development and aging significantly expand the repertoire of bovine miRNAs now shown to be expressed in muscle and could contribute to further studies on growth and developmental disorders in this tissue type.     

HIV associated dementia and HIV encephalitis II: Genes on chromosome 22 expressed in individually microdissected Globus pallidus neurons (Preliminary analysis)

We analyzed RNA gene expression in neurons from 16 cases in four categories, HIV associated dementia with HIV encephalitis (HAD/HIVE), HAD alone, HIVE alone, and HIV-1-positive (HIV+)with neither HAD nor HIVE. We produced the neurons by laser capture microdissection (LCM) from cryopreserved globus pallidus. Of 55,000 gene fragments analyzed, expression of 197 genes was identified with significance (p = 0.005).We examined each gene for its position in the human genome and found a non-stochastic occurrence for only seven genes, on chromosome 22. Six of the seven genes were identified, CSNK1E (casein kinase 1 epsilon), DGCR8 (Di George syndrome critical region 8), GGA1 (Golgi associated gamma adaptin ear containing ARF binding protein 1), MAPK11 (mitogen activated protein kinase 11), SMCR7L (Smith-Magenis syndrome chromosome region candidate 7-like), andTBC1D22A (TBC1 domain family member 22A). Six genes (CSNK1E, DGCR8, GGA1, MAPK11, SMCR7L, and one unidentified gene) had similar expression profiles across HAD/HIVE, HAD, and HIVE vs. HIV+ whereas one gene (TBC1D22A) had a differing gene expression profile across these patient categories. There are several mental disease-related genes including miRNAs on chromosome 22 and two of the genes (DGCR8 and SMCR7L) identified here are mental disease-related. We speculate that dysregulation of gene expression may occur through mechanisms involving chromatin damage and remodeling. We conclude that the pathogenesis of NeuroAIDS involves dysregulation of expression of mental disease-related genes on chromosome 22 as well as additional genes on other chromosomes. The involvement of these genes as well as miRNA requires additional investigation since numerous genes appear to be involved.     

Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.     

Genome-wide identification of Brassica napus microRNAs and their targets in response to cadmium

MicroRNAs (miRNAs) are a distinct class of small RNAs in plants that not only regulate biological processes but also regulate response to environmental stresses. The toxic heavy metal cadmium (Cd) induces expression of several miRNAs in rapeseed (Brassica napus), but it is not known on a genome-wide scale how the expression of miRNAs and their target genes, is regulated by Cd. In this study, four small RNA libraries and four degradome libraries were constructed from Cd-treated and non-Cd-treated roots and shoots of B. napus seedlings. Using high-throughput sequencing, the study identified 84 conserved and non-conserved miRNAs (belonging to 37 miRNA families) from Cd-treated and non-treated B. napus, including 19 miRNA members that were not identified before. Some of the miRNAs were validated by RNA gel blotting. Most of the identified miRNAs were found to be differentially expressed in roots/shoots or regulated by Cd exposure. The study simultaneously identified 802 targets for the 37 (24 conserved and 13 non-conserved) miRNA families, from which there are 200, 537, and 65 targets, belonging to categories I, II, and III, respectively. In category I alone, many novel targets for miRNAs were identified and shown to be involved in plant response to Cd.     

Dysregulated genes and miRNAs in the apoptosis pathway in colorectal cancer patients

Apoptosis is genetically regulated and involves intrinsic and extrinsic pathways. We examined 133 genes within these pathways to identify whether they are expressed differently in colorectal carcinoma (CRC) and normal tissue (N?=?217) and if they are associated with similar differential miRNA expression. Gene expression data (RNA-Seq) and miRNA expression data (Agilent Human miRNA Microarray V19.0) were generated. We focused on dysregulated genes with a fold change (FC) of >?1.50 or <?0.67, that were significant after adjustment for multiple comparisons. miRNA:mRNA seed-region matches were determined. Twenty-three genes were significantly downregulated (FC?<?0.67) and 18 were significantly upregulated (FC?>?1.50). Of these 41 genes, 11 were significantly associated with miRNA differential expression. BIRC5 had the greatest number of miRNA associations (14) and the most miRNAs with a seed-region match (10). Four of these matches, miR-145-5p, miR-150-5p, miR-195-5p, and miR-650, had a negative beta coefficient. CSF2RB was associated with ten total miRNAs (five with a seed-region match, and one miRNA, miR-92a-3p, with a negative beta coefficient). Of the three miRNAs associated with CTSS, miR-20b-5p, and miR-501-3p, had a seed-region match and a negative beta coefficient between miRNA:mRNA pairs. Several miRNAs that were associated with dysregulated gene expression, seed-region matches, and negative beta coefficients also were associated with CRC-specific survival. Our data suggest that miRNAs could influence several apoptosis-related genes. BIRC5, CTSS, and CSF2R all had seed-region matches with miRNAs that would favor apoptosis. Our study identifies several miRNA associated with apoptosis-related genes, that if validated, could be important therapeutic targets.     

MicroRNA signatures associated with immortalization of EBV-transformed lymphoblastoid cell lines and their clinical traits

Objective: MicroRNAs (miRNAs) are negative regulators of gene expression that play important roles in cell processes such as proliferation, development and differentiation. Recently, it has been reported that miRNAs are related to development of carcinogenesis. The aim of this study was to identify miRNAs associated with terminal immortalization of Epstein–Barr virus (EBV)‐transformed lymphoblastoid cell line (LCL) and associated clinical traits. Material and Methods: Hence, we performed miRNA microarray approach with early‐ (p6) and late‐passage (p161) LCLs. Results and Conclusion: Microarray data showed that nine miRNAs (miR‐20b*, miR‐28‐5p, miR‐99a, miR‐125b, miR‐151‐3p, miR‐151:9.1, miR‐216a, miR‐223* and miR‐1296) were differentially expressed in most LCLs during long‐term culture. In particular, miR‐125b was up‐regulated in all the tested late‐passage LCLs. miR‐99a, miR‐125b, miR‐216a and miR‐1296 were putative negative regulators of RASGRP3, GPR160, PRKCH and XAF1, respectively, which were found to be differentially expressed in LCLs during long‐term culture in a previous study. Linear regression analysis showed that miR‐200a and miR‐296‐3p correlated with triglyceride and HbA1C levels, respectively, suggesting that miRNA signatures of LCLs could provide information on the donor’s health. In conclusion, our study suggests that expression changes of specific miRNAs may be required for terminal immortalization of LCLs. Thus, differentially expressed miRNAs would be a potential marker for completion of cell immortalization during EBV‐mediated tumorigenesis.     

Comparison of Microarray Platforms for Measuring Differential MicroRNA Expression in Paired Normal/Cancer Colon Tissues

Background

Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples.

Methodology/Principal Findings

We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport.

Conclusions

Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs – gene expression integration approach.     

Clustered miRNAs and their role in biological functions and diseases

MicroRNAs (miRNAs) are endogenous, small non‐coding RNAs known to regulate expression of protein‐coding genes. A large proportion of miRNAs are highly conserved, localized as clusters in the genome, transcribed together from physically adjacent miRNAs and show similar expression profiles. Since a single miRNA can target multiple genes and miRNA clusters contain multiple miRNAs, it is important to understand their regulation, effects and various biological functions. Like protein‐coding genes, miRNA clusters are also regulated by genetic and epigenetic events. These clusters can potentially regulate every aspect of cellular function including growth, proliferation, differentiation, development, metabolism, infection, immunity, cell death, organellar biogenesis, messenger signalling, DNA repair and self‐renewal, among others. Dysregulation of miRNA clusters leading to altered biological functions is key to the pathogenesis of many diseases including carcinogenesis. Here, we review recent advances in miRNA cluster research and discuss their regulation and biological functions in pathological conditions.     

The Porcine MicroRNA Transcriptome Response to Transmissible Gastroenteritis Virus Infection

Transmissible gastroenteritis virus (TGEV; Coronaviridae family) causes huge economic losses to the swine industry. MicroRNAs (miRNAs) play a regulatory role in viral infection and may be involved in the mammalian immune response. Here, we report a comprehensive analysis of host miRNA expression in TGEV-infected swine testis (ST) cells. Deep sequencing generated 3,704,353 and 2,763,665 reads from uninfected ST cells and infected ST cells, respectively. The reads were aligned to known Sus scrofa pre-miRNAs in miRBase 19, identifying 284 annotated miRNAs. Certain miRNAs were differentially regulated during TGEV infection. 59 unique miRNAs displayed significant differentially expression between the normal and TGEV-infected ST cell samples: 15 miRNAs were significantly up-regulated and 44 were significantly down-regulated. Stem-loop RT-PCR was carried out to determine the expression levels of specific miRNAs in the two samples, and the results were consistent with those of sequencing. Gene ontology enrichment analysis of host target genes demonstrated that the differentially expressed miRNAs are involved in regulatory networks, including cellular process, metabolic process, immune system process. This is the first report of the identification of ST cell miRNAs and the comprehensive analysis of the miRNA regulatory mechanism during TGEV infection, which revealed the miRNA molecular regulatory mechanisms for the viral infection, expression of viral genes and the expression of immune-related genes. The results presented here will aid research on the prevention and treatment of viral diseases.     

The microRNA expression profile in porcine skeletal muscle is changed by constant heat stress

Heat stress has profound effects on animal performance and muscle function, and microRNAs (miRNAs) play a critical role in muscle development and stress responses. To characterize the changes in miRNAs in skeletal muscle responding to heat stress, the miRNA expression profiles of longissimus dorsi muscles of pigs raised under constant heat stress (30 °C; n = 8) or control temperature (22 °C; n = 8) for 21 days were analyzed by Illumina deep sequencing. A total of 58 differentially expressed miRNAs were identified with 30 down‐regulated and 28 up‐regulated, and 63 differentially expressed target genes were predicted by miRNA–mRNA joint analysis. GO and KEGG analyses showed that the genes regulated by differentially expressed miRNAs were enriched in glucose metabolism, cytoskeletal structure and function and stress response. Real‐time PCR showed that the mRNA levels of PDK4, HSP90 and DES were significantly increased, whereas those of SCD and LDHA significantly decreased by heat exposure. The protein levels of CALM1, DES and HIF1α were also significantly increased by constant heat. These results demonstrated that the change in miRNA expression in porcine longissimus dorsi muscle underlies the changes in muscle structure and metabolism in porcine skeletal muscle affected by constant heat stress.     

A transient reversal of miRNA‐mediated repression controls macrophage activation

In mammalian macrophages, the expression of a number of cytokines is regulated by miRNAs. Upon macrophage activation, proinflammatory cytokine mRNAs are translated, although the expression of miRNAs targeting these mRNAs remains largely unaltered. We show that there is a transient reversal of miRNA‐mediated repression during the early phase of the inflammatory response in macrophages, which leads to the protection of cytokine mRNAs from miRNA‐mediated repression. This derepression occurs through Ago2 phosphorylation, which results in its impaired binding to miRNAs and to the corresponding target mRNAs. Macrophages expressing a mutant, non‐phosphorylatable AGO2—which remains bound to miRNAs during macrophage activation—have a weakened inflammatory response and fail to prevent parasite invasion. These findings highlight the relevance of the transient relief of miRNA repression for macrophage function.