Illumina Deep Sequencing Technology Research Articles

Identification and functional prediction of miRNAs that regulate ROS levels in dielectric barrier discharge plasma-treated boar spermatozoa

Dielectric barrier discharge (DBD) plasma regulates the levels of reactive oxygen species (ROS), which are critical for sperm quality. MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules encoded by endogenous genes, which regulate post-transcriptional gene expression in animals. At present, it is unknown whether DBD plasma can regulate sperm ROS levels through miRNAs. To further understand the regulatory mechanism of DBD plasma on sperm ROS levels, miRNAs in fresh boar spermatozoa were detected using Illumina deep sequencing technology. We found that 25 known miRNAs and 50 novel miRNAs were significantly upregulated, and 14 known miRNAs and 74 novel miRNAs were significantly downregulated in DBD plasma-treated spermatozoa. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that target genes of differentially expressed miRNAs were involved in many activities and pathways associated with antioxidants. We verified that DBD plasma significantly increased boar sperm quality and reduced ROS levels. These results suggest that DBD plasma can improve sperm quality by regulating ROS levels via miRNAs. Our findings provide a potential strategy to improve sperm quality through miRNA-targeted regulation of ROS, which helps to increase male reproduction and protect cryopreserved semen in clinical practice.

Differential expression profiles of microRNAs in musk gland of unmated and mated forest musk deer (Moschus berezovskii).

BackgroundThe formation of musk is a complex biophysical and biochemical process that change with the rut of male forest musk deer. We have reported that the mating status of male forest musk deer might result to the variations of chemical composition and microbiota of musk and its yields. Critical roles for microRNAs (miRNAs) of multi-tissues were profiled in our previous study; however, the role for miRNAs of the musk gland remains unclear in this species.MethodsIn this study, we used Illumina deep sequencing technology to sequence the small RNA transcriptome of unmated male (UM) and mated male (UM) of Chinese forest musk deer.ResultsWe identified 1,652 known miRNAs and 45 novel miRNAs, of which there were 174 differentially expressed miRNAs between UM and MM. chi-miR-21-5p, ipu-miR-99b and bta-miR-26a were up-regulated in UM among the 10 most differentially expressed miRNAs. Functional enrichment of the target genes showed that monosaccharide biosynthetic process, protein targeting, cellular protein catabolic process enriched higher in MM. Meanwhile, structural molecule activity, secretion by cell, regulated exocytosis and circulatory system process enriched more in UM, hinting that the formation of musk in UM was mediated by target genes related to exocytosis. The miRNA-mRNA pairs such as miR-21: CHD7, miR143: HSD17B7, miR-141/200a: Noc2 might involve in musk gland development and musk secretion, which need to be verified in future study.

Identification and Characterization of Osmoregulation Related MicroRNAs in Gills of Hybrid Tilapia Under Three Types of Osmotic Stress.

Researchers have increasingly suggested that microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression and protein translation in organs and respond to abiotic and biotic stressors. To understand the function of miRNAs in osmotic stress regulation of the gills of hybrid tilapia (Oreochromis mossambicus ♀ × Oreochromis urolepis hornorum ♂), high-throughput Illumina deep sequencing technology was used to investigate the expression profiles of miRNAs under salinity stress (S, 25‰), alkalinity stress (A, 4‰) and salinity–alkalinity stress (SA, S: 15‰, A: 4‰) challenges. The results showed that 31, 41, and 27 upregulated and 33, 42, and 40 downregulated miRNAs (P < 0.05) were identified in the salt stress, alkali stress, and saline–alkali stress group, respectively, which were compared with those in the control group (C). Fourteen significantly differently expressed miRNAs were selected randomly and then validated by a quantitative polymerase chain reaction. On the basis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, genes related to osmoregulation and biosynthesis were enriched in the three types of osmotic stress. In addition, three miRNAs and three predicted target genes were chosen to conduct a quantitative polymerase chain reaction in the hybrid tilapia and its parents during 96-h osmotic stress. Differential expression patterns of miRNAs provided the basis for research data to further investigate the miRNA-modulating networks in osmoregulation of teleost.

Comparative transcriptome analysis of Salix cupularis under drought stress

Salix cupularis, an afforestation tree for soil and water conservation, is a deciduous willow species commonly cultivated in Sichuan Province, China. The research on the molecular mechanism of drought resistance of S. cupularis will contribute to the subsequent study of soil and water conservation, biodiversity, and ecological protection. Here, the results of an RNA-Seq analysis of transcriptomic data using Illumina deep sequencing technology to compare control (T1) and drought-stressed (T2) seedlings of S. cupularis were reported. Polyethylene glycol was used to induce drought stress in S. cupularis seedlings; 15-day-old seedlings were used for RNA-Seq analysis. De novo gene assembly was used to generate the comprehensive transcriptome, which was comprised of 65,228 unigenes, among which 16,192 were> 1 kb and accounted for 24.82% of the total unigene library. Analysis of the gene structure based on the unigene database, including open reading frame (ORF) predictions, simple sequence repeat (SSR) analysis, and inter-sample single nucleotide polymorphism (SNP) analysis, resulted in the identification of 758,779 SNP and 6779 SSR. After comparing the T1 and T2 gene profiles, 4289 differentially expressed genes (DEGs) were identified, of which 2340 were upregulated and 1949 were downregulated. The gene ontology (GO) analysis classified genes into the cellular component, molecular function, and biological process categories. The clusters of orthologous genes (COG) database was constructed based on the evolutionary phylogeny of bacteria, algae, and eukaryotes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs between T1 and T2 revealed that the top 20 enriched KEGG metabolic pathways were primarily in porphyrin and chlorophyll metabolism, photosynthesis, phenylpropanoid biosynthesis of phenylalanine metabolism, pentose and glucoronate interconversions, and glycan degradation, among other metabolic pathways. Collectively, these findings provide a theoretical basis for future studies on the genetic resistance of S. cupularis, and it also lay a foundation for the flow-up research of soil and water conservation, biodiversity, and ecological protection.

The mitochondrial genome of the dog hookworm Ancylostoma caninum (Nematoda, Ancylostomatidae) from Southwest China

The dog hookworm Ancylostoma caninum (Nematoda, Ancylostomatidae) is a blood-feeding intestinal parasitic nematode and can cause ancylostomiasis in humans. In this study, the complete mitochondrial genome of this anthropozoonotic hookworm was sequenced through Illumina deep sequencing technology. The whole genome was 13,721 bp in length and encoded 36 genes including 12 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. Phylogeny revealed that A. caninum grouped with species from Ancylostomatinae and separated from species of Bunostominae in the family Ancylostomatidae. Amongst the subfamily Ancylostomatinae, three dog-originated A. caninum, regardless of isolate origins, clustered together and were more closely related to the cat hookworm A. tubaeforme and the human hookworm A. duodenale than to the dog/cat hookworm A. ceylanicum and the sea lion hookworm Uncinaria sanguinis. Taken together, the cumulative mitochondrial DNA data provides insights into phylogenetic studies among Ancylostomatidae nematodes.

Profiles analysis reveals circular RNAs involving zebrafish physiological development.

Recent studies have found that known functions of circular RNAs (circRNAs) include sequestration of microRNAs (miRNAs) or proteins, modulation of transcription and interference with splicing, and even translation to produce polypeptides. The zebrafish model is also demonstrably similar to humans in many studies. To explore the changes in circRNAs during embryonic development and to further research the mechanism of action of circRNAs in development-related diseases, Zebrafish embryos at the blastula period, gastrula period, segmentation period, throat stage, and incubation period were collected. Illumina deep-sequencing technology and CircRNA Identifier (CIRI) algorithm were used to detect circRNAs. In total, we identified 1,028 circRNAs (junction reads ≥5 and p < 0.05). Considering that the function of circRNAs is related to host genes, a bioinformatics analysis revealed these differentially expressed host genes are involved in NOTCH signaling pathways, cardiovascular system development, retinal ganglion cell axon guidance, and so on. Moreover, circRNAs can participate in biological regulation through the function of miRNA sponges. TargetScan and miRanda were used to predict 73 miRNAs binding to circRNAs such as miR-19b, miR-124, and so on. Some miRNAs play important roles in embryogenesis. The peak expression of circRNAs is distributed at different time points, suggesting that it may be involved in embryogenesis at different stages. Our study provides a foundation for understanding the dynamic regulation of circRNA transcriptomes during embryogenesis and identifies novel key circRNAs that might control embryonic development in a zebrafish model.

Identification and characterization of miRNAs during flag leaf senescence in rice by high-throughput sequencing

Flag leaf is the last leaf to senesce and its life span plays a critical role in determining grain quality and yield in rice. Little is known about the molecular changes that occur and their regulation in time-dependent manner during flag leaf senescence in rice. Several studies have explored different aspects of miRNA functions in plant development; however, a diminutive account is available about their role in flag leaf senescence. With an aim to unravel the role of miRNAs in ageing of flag leaf in rice, four small RNA libraries were prepared from three stages of senescence and sequenced by Illumina deep sequencing technology. Thirty-eight known and 494 novel miRNAs were identified in the senescing flag leaves. Digital expression analysis revealed that 21 known and 116 novel miRNAs were differentially expressed during senescence. Family member(s) of miR156, miR159, miR160, miR164, miR169, miR171, miR393, miR396, miR535, miR827, miR1428, miR1432 and miR1861 were differentially expressed in at least one stage of flag leaf senescence. The present study has generated a repository of senescence-related miRNAs that can be utilized to contemplate molecular approaches for manipulating the timing of flag leaf senescence in rice and other related crops.

Curcumin may serve an anticancer role in human osteosarcoma cell line U-2 OS by targeting ITPR1.

The present study aimed to determine the mechanisms of action of curcumin in osteosarcoma. Human osteosarcoma U-2 OS cells was purchased from the Cell Bank of the Chinese Academy of Sciences. RNA sequencing analysis was performed for 2 curcumin-treated samples and 2 control samples using Illumina deep sequencing technology. The differentially expressed genes were identified using Cufflink software. Enrichment and protein-protein interaction network analyses were performed separately using cluster Profiler package and Cytoscape software to identify key genes. Then, the mRNA levels of key genes were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in U-2 OS cells. Finally, cell apoptosis, proliferation, migration and invasion arrays were performed. In total, 201 DEGs were identified in the curcumin-treated group. EEF1A1 (degree=88), ATF7IP, HIF1A, SMAD7, CLTC, MCM10, ITPR1, ADAM15, WWP2 and ATP5C1, which were enriched in ‘biological process’, exhibited higher degrees than other genes in the PPI network. RT-qPCR demonstrated that treatment with curcumin was able to significantly increase the levels of CLTC and ITPR1 mRNA in curcumin-treated cells compared with control. In addition, targeting ITPR1 with curcumin significantly promoted apoptosis and suppressed proliferation, migration and invasion. Targeting ITPR1 via curcumin may serve an anticancer role by mediating apoptosis, proliferation, migration and invasion in U-2 OS cells.

Identification of microRNAs and genes associated with hyperandrogenism in the follicular fluid of women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease, which is characterized by hyperandrogenism (HA), chronic anovulation, polycystic ovaries, insulin resistance, and obesity. At present, the mechanism by which PCOS/HA occurs has not been fully elucidated, thus, the mechanisms behind and interventions for HA in PCOS are current hot topics in research. MiRNAs have recently been shown to serve as diagnostic or prognostic biomarkers in patients with cancer. Thus, we are currently focused on studying the altered expression of miRNAs in follicular fluid and their correlation with HA in PCOS. Illumina deep sequencing technology was used to explore different miRNAs in the follicular fluid of women with PCOS/HA and in the follicular fluid of women in a control group. Target prediction databases were then used to analyse the target genes of different expressed miRNAs, and GO analysis and the KEGG pathway database were used to identify the functions and the main biochemical and signalling pathways of differentially expressed target genes. The expression levels of 263 miRNAs were significantly different (>2-fold up-regulated or <0.5-fold down-regulated, P < 0.05) between the two groups of women. For example, the expression levels of miRNA (200a-3p, 10b-3p, 200b-3p, 29c-3p, 99a-3p, and 125a-5p) were significantly increased, while there was a decreased expression of miR-105-3p in PCOS patients with respect to the control. Literature has shown that the above seven miRNAs were associated with HA in PCOS. Furthermore, 31 770 genes were predicted to be targets of the 263 differentially expressed microRNAs. GO analysis and the KEGG pathway database showed involvement of these target genes in HA in PCOS. These results suggest the presence of differentially expressed miRNAs in the follicular fluid of women with PCOS/HA versus women in the control group. The potential role of these microRNAs was elucidated using bioinformatics tools and was found to be involved in the regulation of different pathways, biological functions, and cellular components underlying PCOS. The results of this research may reveal new mechanisms of PCOS/HA and suggest potential treatment targets.

Genome-wide Identification of microRNAs that Respond to Drought Stress in Seedlings of Tertiary Relict Ammopiptanthus mongolicus

microRNAs (miRNAs) are important post-transcriptional regulators of gene expression in plant abiotic stress responses. Ammopiptanthus mongolicus is a typical evergreen woody xerophyte, which makes it an ideal model system for studying drought tolerance in woody plants. The response of miRNAs to drought stress is still unknown in this plant. In this research, we obtained 34 695 665 raw reads from two small RNA libraries constructed from control and drought-treated A. mongolicus seedlings by the Illumina deep sequencing technology. Length analysis revealed that reads of 20, 21, and 24 nucleotides accounted for the majority of the small RNAs in the two libraries. Sequence analyses identified 65 conserved miRNA sequences from 190 members of 31 miRNA families and 20 potential novel miRNAs from 17 families that were differentially expressed between the two libraries. The expression patterns of 25 of these miRNAs were significantly different in the two libraries, but only am-miR4a-c was downregulated during drought stress, the other 24 were upregulated. The expression trends determined by the HiSeq sequencing and by qRT-PCR were similar. Furthermore, 35 target genes for the conserved miRNA were markedly changed and 52 target genes for the potential novel miRNAs were predicted and their functions were annotated by computational analysis. Our results provide new molecular evidence for understanding the molecular mechanisms of drought response and tolerance in A. mongolicus.

Changes of human complexity during maturation: Quantitative assessment on trunk acceleration data

microRNAs (miRNAs) are important post-transcriptional regulators of gene expression in plant abiotic stress responses. Ammopiptanthus mongolicus is a typical evergreen woody xerophyte, which makes it an ideal model system for studying drought tolerance in woody plants. The response of miRNAs to drought stress is still unknown in this plant. In this research, we obtained 34 695 665 raw reads from two small RNA libraries constructed from control and drought-treated A. mongolicus seedlings by the Illumina deep sequencing technology. Length analysis revealed that reads of 20, 21, and 24 nucleotides accounted for the majority of the small RNAs in the two libraries. Sequence analyses identified 65 conserved miRNA sequences from 190 members of 31 miRNA families and 20 potential novel miRNAs from 17 families that were differentially expressed between the two libraries. The expression patterns of 25 of these miRNAs were significantly different in the two libraries, but only am-miR4a-c was downregulated during drought stress, the other 24 were upregulated. The expression trends determined by the HiSeq sequencing and by qRT-PCR were similar. Furthermore, 35 target genes for the conserved miRNA were markedly changed and 52 target genes for the potential novel miRNAs were predicted and their functions were annotated by computational analysis. Our results provide new molecular evidence for understanding the molecular mechanisms of drought response and tolerance in A. mongolicus.

Identification and comparative analysis of the pearl oyster Pinctada fucata hemocytes microRNAs in response to Vibrio alginolyticus infection

MicroRNAs (miRNAs) are a class of noncoding RNA molecules that function as negative regulators of gene expression and play important roles in a wide spectrum of biological processes, including in immune response. However, the physiological regulation function of Pinctada fucata miRNAs, specially their immunomodulation has not been explored yet. Here, two small RNA libraries from hemocytes of P. fucata with or without Vibrio alginolyticus infection were constructed and sequenced using the high-throughput Illumina deep sequencing technology. In total, 11,939,992 and 11,083,327 raw reads, corresponding to 10,993,546 and 9,988,179 clean reads, were respectively obtained in the control and infected libraries. A total of 276 miRNAs, including 225 known miRNAs and 51 putative novel miRNAs, were identified by bioinformatic analysis. By using pairwise comparison between two libraries, 93 miRNAs were found to be significantly differentially expressed, with 42 and 51 miRNAs exhibiting up-regulation and down-regulation, respectively. Thereinto, some known miRNAs were considered to be immune-related. Real-time PCR were implemented for 6 miRNAs co-expressed in the control and infected samples, and agreement was confirmed between the high-throughput sequencing and real-time PCR data. After miRNA targets were predicted, GO and KEGG pathway enrichment analysis were performed, and the results indicated that ten of the differentially expressed miRNAs were involved in immune-related pathways, and might participate in the host immune response to V. alginolyticus. These results of identification and comparative analysis of miRNAs might deepen our understanding of host-pathogen interactions and immune defense mechanisms in P. fucata.

Transcriptome analysis and identification of induced genes in the response of Harmonia axyridis to cold hardiness

Harmonia axyridis is an important predatory lady beetle that is a natural enemy of agricultural and forestry pests. In this research, the cold hardiness induced genes and their expression changes in H. axyridis were screened and detected by the way of the transcriptome and qualitative real-time PCR under normal and low temperatures, using high-throughput transcriptome and digital gene-expression-tag technologies. We obtained a 10Gb transcriptome and an 8Mb gene expression tag pool using Illumina deep sequencing technology and RNA-Seq analysis (accession number SRX540102). Of the 46,980 non-redundant unigenes identified, 28,037 (59.7%) were matched to known genes in GenBank, 21,604 (46.0%) in Swiss-Prot, 19,482 (41.5%) in Kyoto Encyclopedia of Genes and Genomes and 13,193 (28.1%) in Gene Ontology databases. Seventy-five percent of the unigene sequences had top matches with gene sequences from Tribolium castaneum. Results indicated that 60 genes regulated the entire cold-acclimation response, and, of these, seven genes were always up-regulated and five genes always down-regulated. Further screening revealed that six cold-resistant genes, E3 ubiquitin-protein ligase, transketolase, trehalase, serine/arginine repetitive matrix protein 2, glycerol kinase and sugar transporter SWEET1-like, play key roles in the response. Expression from a number of the differentially expressed genes was confirmed with quantitative real-time PCR (HaCS_Trans). The paper attempted to identify cold-resistance response genes, and study the potential mechanism by which cold acclimation enhances the insect's cold endurance. Information on these cold-resistance response genes will improve the development of low-temperature storage technology of natural enemy insects for future use in biological control.

Identification and Characterization of MicroRNAs in the Liver of Blunt Snout Bream (Megalobrama amblycephala) Infected by Aeromonas hydrophila.

MicroRNAs (miRNAs) are small RNA molecules that play key roles in regulation of various biological processes. In order to better understand the biological significance of miRNAs in the context of Aeromonas hydrophila infection in Megalobrama amblycephala, small RNA libraries obtained from fish liver at 0 (non-infection), 4, and 24 h post infection (poi) were sequenced using Illumina deep sequencing technology. A total of 11,244,207, 9,212,958, and 7,939,157 clean reads were obtained from these three RNA libraries, respectively. Bioinformatics analysis identified 171 conserved miRNAs and 62 putative novel miRNAs. The existence of ten randomly selected novel miRNAs was validated by RT-PCR. Pairwise comparison suggested that 61 and 44 miRNAs were differentially expressed at 4 and 24 h poi, respectively. Furthermore, the expression profiles of nine randomly selected miRNAs were validated by qRT-PCR. MicroRNA target prediction, gene ontology (GO) annotation, and Kyoto Encylopedia of Genes and Genomes (KEGG) analysis indicated that a variety of biological pathways could be affected by A. hydrophila infection. Additionally, transferrin (TF) and transferrin receptor (TFR) genes were confirmed to be direct targets of miR-375. These results will expand our knowledge of the role of miRNAs in the immune response of M. amblycephala to A. hydrophila infection, and facilitate the development of effective strategies against A. hydrophila infection in M. amblycephala.

A transcriptomic scan for potential candidate genes involved in osmoregulation in an obligate freshwater palaemonid prawn (Macrobrachium australiense).

BackgroundUnderstanding the genomic basis of osmoregulation (candidate genes and/or molecular mechanisms controlling the phenotype) addresses one of the fundamental questions in evolutionary ecology. Species distributions and adaptive radiations are thought to be controlled by environmental salinity levels, and efficient osmoregulatory (ionic balance) ability is the main mechanism to overcome the problems related to environmental salinity gradients.MethodsTo better understand how osmoregulatory performance in freshwater (FW) crustaceans allow individuals to acclimate and adapt to raised salinity conditions, here we (i), reviewed the literature on genes that have been identified to be associated with osmoregulation in FW crustaceans, and (ii), performed a transcriptomic analysis using cDNA libraries developed from mRNA isolated from three important osmoregulatory tissues (gill, antennal gland, hepatopancreas) and total mRNA from post larvae taken from the freshwater prawn, Macrobrachium australiense using Illumina deep sequencing technology. This species was targeted because it can complete its life cycle totally in freshwater but, like many Macrobrachium sp., can also tolerate brackish water conditions and hence should have genes associated with tolerance of both FW and saline conditions.ResultsWe obtained between 55.4 and 65.2 million Illumina read pairs from four cDNA libraries. Overall, paired end sequences assembled into a total of 125,196 non-redundant contigs (≥200 bp) with an N50 length of 2,282 bp and an average contig length of 968 bp. Transcriptomic analysis of M. australiense identified 32 different gene families that were potentially involved with osmoregulatory capacity. A total of 32,597 transcripts were specified with gene ontology (GO) terms identified on the basis of GO categories. Abundance estimation of expressed genes based on TPM (transcript per million) ≥20 showed 1625 transcripts commonly expressed in all four libraries. Among the top 10 genes expressed in four tissue libraries associated with osmoregulation, arginine kinase and Na+/K+- ATPase showed the highest transcript copy number with 7098 and 660, respectively in gill which is considered to be the most important organ involved in osmoregulation.DiscussionThe current study provides the first broad transcriptome from M. australiense using next generation sequencing and identifies potential candidate genes involved in salinity tolerance and osmoregulation that can provide a foundation for investigating osmoregulatory capacity in a wide variety of freshwater crustaceans.

Identification and characterization of novel and conserved microRNAs in several tissues of the Chinese rare minnow (Gobiocypris rarus) based on illumina deep sequencing technology.

BackgroundMicroRNAs (miRNAs), which comprise a large family of endogenous small non-coding RNA molecules, play important roles in the regulation of gene expression in various biological processes. The Chinese rare minnow (Gobiocypris rarus) is a Chinese native fish species and is used extensively as an experimental fish in China; however, relevant biological data, especially miRNA transcriptome data, have not been well documented. To discover conserved and potential novel miRNAs in Chinese rare minnows, a pool of equal amounts of RNA obtained from 6 different adult rare minnow tissues (brain, eye, gill, liver, muscle and heart) was sequenced using illumina deep sequencing technology.ResultsIn the present study, 26,930,553 raw reads, representing 2,118,439 unique high-quality reads, were obtained from the pooled small RNA library. Using bioinformatics analysis, 352 conserved and 112 novel Chinese rare minnow miRNAs were first discovered and characterized in this study. Moreover, we found extensive sequence variations (isomiRs) in rare minnow miRNAs, including internal miRNA isomiRs and terminal isomiRs at both the 5′ and 3′ ends and nucleotide variants. Six conserved and 4 novel miRNAs were selected and validated in 6 different adult rare minnow tissues using quantitative real-time PCR (qPCR). The results showed that miR-30a, miR-30b, and Novel-37 are ubiquitously expressed in a variety of tissues. miR-16a, miR-9, miR-125b, miR-34a, and Novel-69 were predominantly expressed in the brain. Novel-115 and Novel-7 were highly expressed in gills, but were relatively weakly expressed in other tissues. These results provided the expression patterns of miRNA genes in Chinese rare minnow. Finally, based on bioinformatics predictions, we mainly found that Novel-94 and Novel-1b-5p were simultaneously targeted to the 3′UTR of Dmrt1, which controls sex determination and/or sexual differentiation in a variety of metazoans at different sites. Novel-29b targeted the 3′UTR of Foxl2, which is involved in the maintenance of ovarian function and the transcriptional regulation of gonadal differentiation-related genes. Novel-62 and Novel-53 targeted the 3′UTR of ERbeta1 and ERbeta2 (which regulate the transcription of target genes), respectively.ConclusionsRare minnow is a widely used model for assessing the risk of environmental pollution in China. Identifying and characterizing rare minnow miRNA genes is necessary to discover the biological function of miRNAs and to screen for new molecule biomarkers to assess the risk of environmental pollution in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2606-5) contains supplementary material, which is available to authorized users.

MicroRNA-dependent gene regulatory networks in maize leaf senescence.

BackgroundMaize grain yield depends mainly on the photosynthetic efficiency of functional leaves, which is controlled by an array of gene networks and other factors, including environmental conditions. MicroRNAs (miRNAs) are small RNA molecules that play important roles in plant developmental regulation. A few senescence-associated miRNAs (SA-miRNAs) have been identified as important participants in regulating leaf senescence by modulating the expression levels of their target genes.ResultsTo elucidate miRNA roles in leaf senescence and their underlying molecular mechanisms in maize, a stay-green line, Yu87-1, and an early leaf senescence line, Early leaf senescence-1 (ELS-1), were selected as experimental materials for the differential expression of candidate miRNAs. Four small RNA libraries were constructed from ear leaves at 20 and 30 days after pollination and sequenced by Illumina deep sequencing technology. Altogether, 81 miRNAs were detected in both lines. Of these, 16 miRNAs of nine families were differentially expressed between ELS-1 andYu87-1. The phenotypic and chlorophyll content analyses of both lines identified these 16 differentially expressed miRNAs as candidate SA-miRNAs.ConclusionsIn this study, 16 candidate SA-miRNAs of ELS-1 were identified through small RNA deep sequencing technology. Degradome sequencing results indicated that these candidate SA-miRNAs may regulate leaf senescence through their target genes, mainly transcription factors, and potentially control chlorophyll degradation pathways. The results highlight the regulatory roles of miRNAs during leaf senescence in maize.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0755-y) contains supplementary material, which is available to authorized users.

Characterization of microRNAs and their targets in wild barley (Hordeum vulgare subsp. spontaneum) using deep sequencing.

MicroRNAs (miRNA) are a class of small, endogenous RNAs that play a negative regulatory role in various developmental and metabolic processes of plants. Wild barley (Hordeum vulgare subsp. spontaneum), as the progenitor of cultivated barley (Hordeum vulgare subsp. vulgare), has served as a valuable germplasm resource for barley genetic improvement. To survey miRNAs in wild barley, we sequenced the small RNA library prepared from wild barley using the Illumina deep sequencing technology. A total of 70 known miRNAs and 18 putative novel miRNAs were identified. Sequence analysis revealed that all of the miRNAs identified in wild barley contained the highly conserved hairpin sequences found in barley cultivars. MiRNA target predictions showed that 12 out of 52 miRNA families were predicted to target transcription factors, including 8 highly conserved miRNA families in plants and 4 wheat-barley conserved miRNA families. In addition to transcription factors, other predicted target genes were involved in diverse physiological and metabolic processes and stress defense. Our study for the first time reported the large-scale investigation of small RNAs in wild barley, which will provide essential information for understanding the regulatory role of miRNAs in wild barley and also shed light on future practical utilization of miRNAs for barley improvement.

MicroRNAs of the mesothorax in Qinlingacris elaeodes, an alpine grasshopper showing a wing polymorphism with unilateral wing form.

MicroRNAs (miRNAs) are now recognized as key post-transcriptional regulators in regulation of phenotypic diversity. Qinlingacris elaeodes is a species of the alpine grasshopper, which is endemic to China. Adult individuals have three wing forms: wingless, unilateral-winged and short-winged. This is an ideal species to investigate the phenotypic plasticity, development and evolution of insect wings because of its case of unilateral wing form in both the sexes. We sequenced a small RNA library prepared from mesothoraxes of the adult grasshoppers using the Illumina deep sequencing technology. Approximately 12,792,458 raw reads were generated, of which the 854,580 high-quality reads were used only for miRNA identification. In this study, we identified 49 conserved miRNAs belonging to 41 families and 69 species-specific miRNAs. Moreover, seven miRNA*s were detected both for conserved miRNAs and species-specific miRNAs, which were supported by hairpin forming precursors based on polymerase chain reaction. This is the first description of miRNAs in alpine grasshoppers. The results provide a useful resource for further studies on molecular regulation and evolution of miRNAs in grasshoppers. These findings not only enrich the miRNAs for insects but also lay the groundwork for the study of post-transcriptional regulation of wing forms.

MiR-34a screened by miRNA profiling negatively regulates Wnt/β-catenin signaling pathway in Aflatoxin B1 induced hepatotoxicity.

Aflatoxin-B1 (AFB1), a hepatocarcinogenic mycotoxin, was demonstrated to induce the high rate of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) participate in the regulation of several biological processes in HCC. However, the function of miRNAs in AFB1-induced HCC has received a little attention. Here, we applied Illumina deep sequencing technology for high-throughout profiling of microRNAs in HepG2 cells lines after treatment with AFB1. Analysis of the differential expression profile of miRNAs in two libraries, we identified 9 known miRNAs and 1 novel miRNA which exhibited abnormal expression. KEGG analysis indicated that predicted target genes of differentially expressed miRNAs are involved in cancer-related pathways. Down-regulated of Drosha, DGCR8 and Dicer 1 indicated an impairment of miRNA biogenesis in response to AFB1. miR-34a was up-regulated significantly, down-regulating the expression of Wnt/β-catenin signaling pathway by target gene β-catenin. Anti-miR-34a can significantly relieved the down-regulated β-catenin and its downstream genes, c-myc and Cyclin D1, and the S-phase arrest in cell cycle induced by AFB1 can also be relieved. These results suggested that AFB1 might down-regulate Wnt/β-catenin signaling pathway in HepG2 cells by up-regulating miR-34a, which may involve in the mechanism of liver tumorigenesis.