Characteristics of conserved microRNAome and their evolutionary adaptation to regulation of immune defense functions in the spleen of silver carp and bighead carp.

Immune defense functions of silver carp (Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichthys nobilis) have shown obvious evolutionary divergence. MiRNAs participate in the fine regulation of immune function. However, the evolutionary adaptation of miRNAs in the regulation of immune defense function is still poorly understood in silver carp and bighead carp. Here, small RNA libraries were constructed from the spleen tissue of one-year-old and three-year-old healthy silver carp and bighead carp, 424 and 422 known conserved miRNAs were respectively identified from the spleen of silver carp and bighead carp by bioinformatic analysis, which 398 were shared between the two species. These conserved miRNAs showed highly similar expression patterns between silver carp and bighead carp, but the abundance in spleen varied greatly in different species. Family analysis showed that miRNA families including mir-8, mir-7, mir-23, mir-338, mir-30, mir-27, mir-221, mir-19, mir-181, mir-17, mir-15, mir-148, mir-130, mir-10 and let-7 were the main miRNAs in the spleen of silver carp and bighead carp. 27 and 51 significant differentially expressed (SDE) miRNAs were identified from silver carp and bighead carp, respectively. Evolution analysis for the predicted target genes of SDE-miRNAs showed that ten biological processes such as blood coagulation, cell adhesion mediated by integrin and adaptive immune response were positively selected. In addition, immune genes including TLR3, NFATC3, MALT1, B2M, GILT and MHCII were positively selected only in silver carp, and they were specifically targeted by the SDE-miRNAs including miR-9-5p, miR-196a-5p, miR-375, miR-122, miR-722, miR-132-3p, miR-727-5p, miR-724, miR-19d-5p and miR-138-5p, respectively. PLA2G4 in Fc epsilon RI signaling pathway was positively selected only in bighead carp and was specifically targeted by the SDE-miRNAs including miR-222b, miR-22b-5p, miR-15c, miR-146a, miR-125c-3p, miR-221-5p, miR-2188-5p, miR-142a-3p, miR-212, miR-138-5p and miR-15b-5p. In particular, SDE-miRNAs such as miR-144-3p, miR-2188-3p, miR-731, miR-363-3p and miR-218b could simultaneously target multiple evolutionarily differentiated immune-related genes. These results indicated that in the spleen of silver carp and bighead carp, conserved miRNAs have obvious evolutionary adaptations in the regulation of immune defense function. The results of this study can provide valuable resources for further revealing themechanism of miRNA in the formation of resistance traits evolution between silver carp and bighead carp.

Exploring the Integrated Role of miRNAs and lncRNAs in Regulating the Transcriptional Response to Amino Acids and Insulin-like Growth Factor 1 in Gilthead Sea Bream (Sparus aurata) Myoblasts.

In this study, gilthead sea bream (Sparus aurata) fast muscle myoblasts were stimulated with two pro-growth treatments, amino acids (AA) and insulin-like growth factor 1 (Igf-1), to analyze the transcriptional response of mRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and to explore their possible regulatory network using bioinformatic approaches. AA had a higher impact on transcription (1795 mRNAs changed) compared to Igf-1 (385 mRNAs changed). Both treatments stimulated the transcription of mRNAs related to muscle differentiation (GO:0042692) and sarcomere (GO:0030017), while AA strongly stimulated DNA replication and cell division (GO:0007049). Both pro-growth treatments altered the transcription of over 100 miRNAs, including muscle-specific miRNAs (myomiRs), such as miR-133a/b, miR-206, miR-499, miR-1, and miR-27a. Among 111 detected lncRNAs (>1 FPKM), only 30 were significantly changed by AA and 11 by Igf-1. Eight lncRNAs exhibited strong negative correlations with several mRNAs, suggesting a possible regulation, while 30 lncRNAs showed strong correlations and interactions with several miRNAs, suggesting a role as sponges. This work is the first step in the identification of the ncRNAs network controlling muscle development and growth in gilthead sea bream, pointing out potential regulatory mechanisms in response to pro-growth signals.

circSMARCA5 Is an Upstream Regulator of the Expression of miR-126-3p, miR-515-5p, and Their mRNA Targets, Insulin-like Growth Factor Binding Protein 2 (IGFBP2) and NRAS Proto-Oncogene, GTPase (NRAS) in Glioblastoma.

The involvement of non-coding RNAs (ncRNAs) in glioblastoma multiforme (GBM) pathogenesis and progression has been ascertained but their cross-talk within GBM cells remains elusive. We previously demonstrated the role of circSMARCA5 as a tumor suppressor (TS) in GBM. In this paper, we explore the involvement of circSMARCA5 in the control of microRNA (miRNA) expression in GBM. By using TaqMan® low-density arrays, the expression of 748 miRNAs was assayed in U87MG overexpressing circSMARCA5. Differentially expressed (DE) miRNAs were validated through single TaqMan® assays in: (i) U87MG overexpressing circSMARCA5; (ii) four additional GBM cell lines (A172; CAS-1; SNB-19; U251MG); (iii) thirty-eight GBM biopsies; (iv) twenty biopsies of unaffected brain parenchyma (UC). Validated targets of DE miRNAs were selected from the databases TarBase and miRTarbase, and the literature; their expression was inferred from the GBM TCGA dataset. Expression was assayed in U87MG overexpressing circSMARCA5, GBM cell lines, and biopsies through real-time PCR. TS miRNAs 126-3p and 515-5p were upregulated following circSMARCA5 overexpression in U87MG and their expression was positively correlated with that of circSMARCA5 (r-values = 0.49 and 0.50, p-values = 9 × 10-5 and 7 × 10-5, respectively) in GBM biopsies. Among targets, IGFBP2 (target of miR-126-3p) and NRAS (target of miR-515-5p) mRNAs were positively correlated (r-value = 0.46, p-value = 0.00027), while their expression was negatively correlated with that of circSMARCA5 (r-values = -0.58 and -0.30, p-values = 0 and 0.019, respectively), miR-126-3p (r-value = -0.36, p-value = 0.0066), and miR-515-5p (r-value = -0.34, p-value = 0.010), respectively. Our data identified a new GBM subnetwork controlled by circSMARCA5, which regulates downstream miRNAs 126-3p and 515-5p, and their mRNA targets IGFBP2 and NRAS.

A Neutralizing Aptamer to TGFBR2 and miR-145 Antagonism Rescue Cigarette Smoke- and TGF-ß-Mediated CFTR Expression.

Transforming growth factor ß (TGF-ß), signaling induced by cigarette smoke (CS), plays an important role in the progression of airway diseases, like chronic bronchitis associated with chronic obstructive pulmonary disease (COPD), and in smokers. Chronic bronchitis is characterized by reduced mucociliary clearance (MCC). Cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in normal MCC. TGF-ß and CS (via TGF-ß) promote acquired CFTR dysfunction by suppressing CFTR biogenesis and function. Understanding the mechanism by which CS promotes CFTR dysfunction can identify therapeutic leads to reverse CFTR suppression and rescue MCC. TGF-ß alters the microRNAome of primary human bronchial epithelium. TGF-ß and CS upregulate miR-145-5p expression to suppress CFTR and the CFTR modifier, SLC26A9. miR-145-5p upregulation with a concomitant CFTR and SLC26A9 suppression was validated in CS-exposed mouse models. While miR-145-5p antagonism rescued the effects of TGF-ß in bronchial epithelial cells following transfection, an aptamer to block TGF-ß signaling rescues CS- and TGF-ß-mediated suppression of CFTR biogenesis and function in the absence of any transfection reagent. These results demonstrate that miR-145-5p plays a significant role in acquired CFTR dysfunction by CS, and they validate a clinically feasible strategy for delivery by inhalation to locally modulate TGF-ß signaling in the airway and rescue CFTR biogenesis and function.

Integrative analysis of the Pekin duck (Anas anas) MicroRNAome during feather follicle development.

BACKGROUND: The quality and yield of duck feathers are very important economic traits that might be controlled by miRNA regulation. The aim of the present study was to investigate the mechanism underlying the crosstalk between individual miRNAs and the activity of signaling pathways that control the growth of duck feathers during different periods. We therefore conducted a comprehensive investigation using Solexa sequencing technology on the Pekin duck microRNAome over six stages of feather development at days 11, 15, and 20 of embryonic development (during the hatching period), and at 1 day and 4 and 10 weeks posthatch. RESULTS: There were a total of 354 known miRNAs and 129 novel candidate miRNAs found based on comparisons with known miRNAs in the Gallus gallus miRBase. The series of miRNAs related to feather follicle formation as summarized in the present study showed two expression patterns, with primary follicle developed during embryonic stage and secondary follicle developed mainly at early post hatch stage. Analysis of miRNA expression profiles identified 18 highly expressed miRNAs, which might be directly responsible for regulation of feather development. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that in addition to Wnt and transforming growth factor (TGFß) signaling pathways, which were widely reported in response to follicle formation, another group of signaling pathways that regulate lipid synthesis and metabolism, such as the phosphatidylinositol signaling system and glycerolipid metabolism and signaling, are also responsible for follicle formation. CONCLUSION: The highly expressed miRNAs provide a valuable reference for further investigation into the functional miRNAs important for feather development. Lipid synthesis and metabolism related signaling pathways might be responsible for lipid formation on the surface of feather, and should be paid much more attention for their relation to feather quality.

Dynamic microRNAome profiles in the developing porcine liver.

Increasing evidence suggests that micro (mi)RNAs play important roles in various biological process. To evaluate the roles of miRNA in the porcine liver, we investigated the dynamic profiles of microRNAomes using liver tissue from pigs during the embryonic period (embryonic day 90), weaning stage (postnatal day 30), and adult stage (7 years old). A total of 186 unique miRNAs were differentially expressed during liver development. We also identified that 17, 13, and 6 miRNAs were specifically abundant at embryonic day 90, postnatal day 30, and at 7 years, respectively. Besides regulating basic cellular roles in development, miRNAs expressed at the three developmental stages also participated in regulating "embryonic liver development," "early hepatic growth and generating a functioning liver," and "energy metabolic processes," respectively. Our study indicates that miRNAs are extensively involved in liver development, and provides a valuable resource for the further elucidation of miRNA regulatory roles during liver development.

Evolutionary suppression of erythropoiesis via the modulation of TGF-ß signalling in an Antarctic icefish.

The Antarctic icefish, a family (Channichthyidae) of teleosts within the perciform suborder Notothenioidei, are the only known vertebrates without oxygen-transporting haemoglobins and that are largely devoid of circulating erythrocytes. To elucidate the evo-devo mechanisms underpinning the suppressed erythropoiesis in the icefish, we conducted comparative studies on the transcriptomes and microRNAomes of the primary haematopoietic tissues between an icefish (Chionodraco hamatus) and two red-blooded notothenioids (Trematomus bernacchii and Gymnodraco acuticeps). We identified substantial remodelling of the haematopoietic programs in the icefish through which erythropoiesis is selectively suppressed. Experimental verification showed that erythropoietic suppression in the icefish may be attributable to the upregulation of TGF-ß signalling, which coincides with reductions in multiple transcription factors essential for erythropoiesis and the upregulation of hundreds of microRNAs, the majority (> 80%) of which potentially target erythropoiesis regulating factors. Of the six microRNAs selected for verification, three miRNAs (miR-152, miR-1388 and miR-16b) demonstrated suppressive functions on GATA1 and ALAS2, which are two factors important for erythroid differentiation, resulting in reduced numbers of erythroids in microinjected zebra fish embryos. Codon substitution analyses of the genes of the TGF-ß superfamily revealed signs of positive selection in TGF-ß1 and endoglin in the lineages leading to Antarctic notothenioids. Both genes are previously known to function in erythropoietic suppression. These findings implied a general trend of erythropoietic suppression in the cold-adapted notothenioid lineages through evolutionary modulation of the multi-functional TGF-ß signalling pathway. This trend is more pronounced in the haemoglobin-less icefish, which may pre-emptively hinder the otherwise defective erythroids from production.

MicroRNAs as Candidate Biomarkers for Alzheimer's Disease.

The neurological damage of Alzheimer's disease (AD) is thought to be irreversible upon onset of dementia-like symptoms, as it takes years to decades for occult pathologic changes to become symptomatic. It is thus necessary to identify individuals at risk for the development of the disease before symptoms manifest in order to provide early intervention. Surrogate markers are critical for early disease detection, stratification of patients in clinical trials, prediction of disease progression, evaluation of response to treatment, and also insight into pathomechanisms. Here, we review the evidence for a number of microRNAs that may serve as biomarkers with possible mechanistic insights into the AD pathophysiologic processes, years before the clinical manifestation of the disease.

MicroRNA and Other Non-Coding RNAs in Epstein-Barr Virus-Associated Cancers.

EBV is a direct causative agent in around 1.5% of all cancers. The oncogenic properties of EBV are related to its ability to activate processes needed for cellular proliferation, survival, migration, and immune evasion. The EBV latency program is required for the immortalization of infected B cells and involves the expression of non-coding RNAs (ncRNAs), including viral microRNAs. These ncRNAs have different functions that contribute to virus persistence in the asymptomatic host and to the development of EBV-associated cancers. In this review, we discuss the function and potential clinical utility of EBV microRNAs and other ncRNAs in EBV-associated malignancies. This review is not intended to be comprehensive, but rather to provide examples of the importance of ncRNAs.

Regulation of rumen development in neonatal ruminants through microbial metagenomes and host transcriptomes.

BACKGROUND: In ruminants, early rumen development is vital for efficient fermentation that converts plant materials to human edible food such as milk and meat. Here, we investigate the extent and functional basis of host-microbial interactions regulating rumen development during the first 6 weeks of life. RESULTS: The use of microbial metagenomics, together with quantification of volatile fatty acids (VFAs) and qPCR, reveals the colonization of an active bacterial community in the rumen at birth. Colonization of active complex carbohydrate fermenters and archaea with methyl-coenzyme M reductase activity was also observed from the first week of life in the absence of a solid diet. Integrating microbial metagenomics and host transcriptomics reveals only 26.3% of mRNA transcripts, and 46.4% of miRNAs were responsive to VFAs, while others were ontogenic. Among these, one host gene module was positively associated with VFAs, while two other host gene modules and one miRNA module were negatively associated with VFAs. Eight host genes and five miRNAs involved in zinc ion binding-related transcriptional regulation were associated with a rumen bacterial cluster consisting of Prevotella, Bacteroides, and Ruminococcus. CONCLUSION: This three-way interaction suggests a potential role of bacteria-driven transcriptional regulation in early rumen development via miRNAs. Our results reveal a highly active early microbiome that regulates rumen development of neonatal calves at the cellular level, and miRNAs may coordinate these host-microbial interactions.

Comparative phenotype and microRNAome in developing anthers of wild-type and male-sterile Lycium barbarum L.

Lycium barbarum L. (L. barbarum) is an economically important plant, as its fruit is highly marketable for its healthy nutrient content. In this study, we characterized the anther development of a major cultivar (Ningqi No. 1) and a male-sterile mutant (Ningqi No. 5) of L. barbarum. We initially investigated the phenotypes of Ningqi No. 1 and Ningqi No. 5 using microscopy and chemical staining, which showed that Ningqi No. 5 failed in the degradation of anther callose, leading to an absence of mature pollen grains and thus to male sterility. Then, to understand the dynamic profile of miRNA expression during the development of the anthers, we collected anther samples from both Ningqi No. 1 and Ningqi No. 5 throughout anther development, and we further identified 137 novel miRNAs from these anther samples by using next-generation deep sequencing technology. Of these 137 novel miRNAs, 96 miRNAs were conserved miRNAs classified into 65 miRNA families, including a few well-known miRNA families related to anther development, such as miR156, miR159 and miR172. In addition, the remaining 41 miRNAs were considered lineage-specific miRNAs, which had no orthologues in other species. The expression data showed that 45 of the 137 miRNAs were differentially expressed in the different samples, including 4 Ningqi No. 5-specific miRNAs and 15 stage-specific miRNAs. The expression patterns of six miRNAs and their predicted targets were verified by Q-PCR, and one of miRNAs and its target were chosen for transient co-expression in Nicotiana benthamiana leaves to verify the correlations between the miRNA and its predicted target. Overall, the identification of the miRNAs in the anther development of Ningqi No. 1 and Ningqi No. 5 provides a valuable resource for understanding the molecular mechanisms of male sterility in L. barbarum.

Integration of microRNAome, proteomics and metabolomics to analyze arsenic-induced malignant cell transformation.

Long-term exposure to arsenic has been linked to tumorigenesis in different organs and tissues, such as skin; however, the detailed mechanism remains unclear. In this present study, we integrated "omics" including microRNAome, proteomics and metabolomics to investigate the potential molecular mechanisms. Compared with non-malignant human keratinocytes (HaCaT), twenty-six miRNAs were significantly altered in arsenic-induced transformed cells. Among these miRNAs, the differential expression of six miRNAs was confirmed using Q-RT-PCR, representing potential oxidative stress genes. Two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry (MS) were performed to identify the differential expression of proteins in arsenic-induced transformed cells, and twelve proteins were significantly changed. Several proteins were associated with oxidative stress and carcinogenesis including heat shock protein beta-1 (HSPB1), peroxiredoxin-2 (PRDX2). Using ultra-performance liquid chromatography and Q-TOF mass spectrometry (UPLC/Q-TOF MS), 68 metabolites including glutathione, fumaric acid, citric acid, phenylalanine, and tyrosine, related to redox metabolism, glutathione metabolism, citrate cycle, met cycle, phenylalanine and tyrosine metabolism were identified and quantified. Taken together, these results indicated that arsenic-induced transformed cells exhibit alterations in miRNA, protein and metabolite profiles providing novel insights into arsenic-induced cell malignant transformation and identifying early potential biomarkers for cutaneous squamous cell carcinoma induced by arsenic.

Low dose irradiation profoundly affects transcriptome and microRNAme in rat mammary gland tissues.

Ionizing radiation has been successfully used in medical tests and treatment therapies for a variety of medical conditions. However, patients and health-care workers are greatly concerned about overexposure to medical ionizing radiation and possible cancer induction due to frequent mammographies and/or CT scans. Diagnostic imaging involves the use of low doses of ionizing radiation, and its potential carcinogenic role creates a cancer risk concern for exposed individuals. In this study, the effects of X-ray exposure of different doses on the gene expression patterns and the micro-RNA expression patterns in normal breast tissue were investigated in rats. Our results revealed the activation of immune response pathways upon low dose of radiation exposure. These included natural killer mediated cytotoxicity pathways, antigen processing and presentation pathways, chemokine signaling pathways, and T- and B-cell receptor signaling pathways. Both high and low doses of radiation led to miRNA expression alterations. Increased expression of miR-34a may be linked to cell cycle arrest and apoptosis. Up-regulation of miR-34a was correlated with down-regulation of its target E2F3 and up-regulation of p53. This data suggests that ionizing radiation at specific high and low doses leads to cell cycle arrest and a possible initiation of apoptosis.