Solexa Sequencing Research Articles

MiR-21 regulated hair follicle cycle development in Cashmere goats by targeting FGF18 and SMAD7

Increasing Cashmere production can add value because it is the primary product of Cashmere goats. Recent years, peoples find miRNAs are crucial in regulating the development of hair follicle. Following Solexa sequencing, many miRNAs were distinguishingly expressed in telogen skin samples of goats and sheep in earlier study. But the method through which miR-21 controls the growth of hair follicles is still ambiguous. Bioinformatics analysis was used to predict the target genes of miR-21. The mRNA level of miR-21 in telogen Cashmere goat skins was higher than in anagen, according to the results of qRT-PCR, and the target genes expressed similarly with miR-21. Western blot showed similar trend, the protein expression of FGF18 and SMAD7 were lower in anagen samples. The Dual-Luciferase reporter assay confirmed miRNA-21's relationship with its target gene, and the consequences indicated found FGF18 and SMAD7 have positive correlations with miR-21. Western blot and qRT-PCR distinguished the expression of protein and mRNA in miR-21 and its target genes. According to the consequence, we found that target genes expression was increased by miR-21 in HaCaT cells. This study identified that miR-21 might take part in the development of Cashmere goat’s hair follicles by targeting FGF18 and SMAD7.

Expression characteristics of piRNAs in ovine luteal phase and follicular phase ovaries.

PIWI-interacting RNAs (piRNAs), as a novel class of small non-coding RNAs that have been shown to be indispensable in germline integrity and stem cell development. However, the expressed characteristics and regulatory roles of piRNAs during different reproductive phases of animals remain unknown. In this study, we investigated the piRNAs expression profiles in ovaries of sheep during the luteal phase (LP) and follicular phase (FP) using the Solexa sequencing technique. A total of 85,219 and 1,27,156 piRNAs tags were identified in ovine ovaries across the two phases. Most expressed piRNAs start with uracil. piRNAs with a length of 24 nt or 27–29 nts accounted for the largest proportion. The obvious ping-pong signature appeared in the FP ovary. The piRNA clusters in the sheep ovary were unevenly distributed on the chromosomes, with high density on Chr 3 and 1. For genome distribution, piRNAs in sheep ovary were mainly derived from intron, CDS, and repeat sequence regions. Compared to the LP ovary, a greater number of expressed piRNA clusters were detected in the FP ovary. Simultaneously, we identified 271 differentially expressed (DE) piRNAs between LP and FP ovaries, with 96 piRNAs upregulated and 175 piRNAs downregulated, respectively. Functional enrichment analysis (GO and KEGG) indicated that their target genes were enriched in reproduction-related pathways including oocyte meiosis, PI3K-Akt, Wnt, and TGF-β signaling pathways. Together, our results highlighted the sequence and expression characteristics of the piRNAs in the sheep ovary, which will help us understand the roles of piRNAs in the ovine estrus cycle.

Characterization of MicroRNAs Associated with Reproduction in the Brown Planthopper, Nilaparvata lugens.

Insects have a robust capacity to produce offspring for propagation, and the reproductive events of female insects have been achieved at the molecular and physiological levels via regulatory gene pathways. However, the roles of MicroRNAs (miRNAs) in the reproductive development of the brown planthopper (BPH), Nilaparvata lugens, remain largely unexplored. To understand the roles of miRNAs in reproductive development, miRNAs were identified by Solexa sequencing in short-winged (SW) female adults of BPH. Small RNA libraries derived from three developmental phases (1 day, 3 days, and 5 days after emergence) were constructed and sequenced. We identified 905 miRNAs, including 263 known and 642 novel miRNAs. Among them, a total of 43 miRNAs were differentially expressed in the three developmental phases, and 14,568 putative targets for 43 differentially expressed miRNAs (DEMs) were predicted by TargetScan and miRanda. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the predicted miRNA targets illustrated the putative roles for these DEMs in reproduction. The progress events were annotated, including oogenesis, lipid biosynthetic process, and related pathways such as apoptosis, ABC transporters, and amino acid metabolism. Four highly abundant DEMs (miR-9a-5p, miR-34-5p, miR-275-3p, and miR-317-3p) were further screened, and miR-34-5p was confirmed to be involved in the regulation of reproduction. Overexpression of miR-34-5p via injecting its mimics reduced fecundity and decreased Vg expression. Moreover, target genes prediction for miR-34-5p showed they might be involved in 20E signaling cascades, apoptosis, and gonadal development, including hormone receptor 4 (HR4), caspase-1 (Cp-1), and spermatogenesis-associated protein 20 (SPATA20). These findings provide a valuable resource for future studies on the role of miRNAs in BPH reproductive development.

Genome-wide analysis of sucrose synthase family in soybean and their expression in response to abiotic stress and seed development

The sucrose synthase (SS) is an important enzyme family which play a vital role in sugar metabolism to improve the fruit quality of the plants. In many plant species, the members of SS family have been investigated but the detailed information is not available in legumes particularly and Glycine max specifically. In the present study, we found thirteen SS members (GmSS1-GmSS13) in G. max genome. High conserved regions were present in the GmSS sequences that may due to the selection pressure during evolutionary events. The segmental duplication was the major factor to increase the number of GmSS family members. The identified thirteen GmSS genes were divided into Class I, Class II and Class III with variable numbers of genes in each class. The protein interaction of GmSS gave the co-expression of sucrose synthase with glucose-1-phosphate adenylyltransferase while SLAC and REL test found number of positive sites in the coding sequences of SS family members. All the GmSS family members except GmSS7 and few of class III members, were highly expressed in all the soybean tissues. The expression of the class I members decreased during seed development, whireas, the class II members expression increased during the seed developing, may involve in sugar metabolism during seed development. Solexa sequencing libraries of acidic condition (pH 4.2) stress samples showed that the expression of class I GmSS genes increased 1- to 2-folds in treated samples than control. The differential expression pattern was observed between the members of a paralogous. This study provides detailed genome-wide analysis of GmSS family in soybean that will provide new insights for future evolutionary and soybean breeding to improve the plant growth and development.

MiR-19 3b regulated the formation of coat colors by targeting WNT10A and GNAI2 in Cashmere goats

MiRNAs as a series of small noncoding RNAs that play a crucial part in regulating coat color and hair follicle development. In the previous Solexa sequencing experiments, there were many miRNAs expressed differentially in alpacas with different coat color, including miR-193b.But the mechanism of miR-193b in mammalian pigmentation is still unknown. In this study, bioinformatics analysis showed that WNT10A and GNAI2 might be the target genes of miR-193b. qRT-PCR showed the expression of miR-193b in white Cashmere goats’ skins was obviously lower than that in browns, and the expression of WNT10A and GNAI2 were similar with miR-193b. The protein levels of WNT10A and GNAI2 indicated the same findings. Furthermore, the expression of WNT10A and GNAI2 in keratinocytes were analyzed from mRNA and protein levels, the results manifested that the group of overexpression of miR-193b in HaCaT cells increased the expressions of target genes, and miR-193b inhibition group reduced expressions. Luciferase report assays confirmed that the targeting relationship between miR-193b and target genes (WNT10A and GNAI2), the results showed that miR-193b was positively correlated with target genes. These experimental data showed that miR-193b might participate in adjustment of coat color in skin tissue of Cashmere goat by targeting WNT10A and GNAI2.

Changes of Intestinal Flora and Lymphocyte Subsets in Patients with Chronic Renal Failure.

Objective To explore the changes of intestinal flora and lymphocyte subsets in patients with chronic renal failure (CRF). Methods 60 CRF patients who were treated from June 2018 to June 2019 were selected; 60 healthy persons were selected as the control group. 16S rDNA was used to detect the expression of Lactobacillus, Enterobacteriaceae, Enterococcus, Bacteroides, Clostridium, and Bifidobacterium in the feces of the two groups. Illumina Miseq sequencing (Solexa sequencing technology) method was used to analyze the structural differences and species diversity of intestinal flora, including species richness index (Chao) and diversity index (Shannon, Simpson). Flow cytometry was used to detect the levels of lymphocytes and their subgroups of the two groups. Pearson correlation analysis was used to analyze the correlation between Chao and lymphocyte subsets. Results The number of Enterobacteriaceae and Enterococcus in CRF group were higher than those in the control group (P < 0.05), while the Lactobacillus, Bacteroides, Clostridium, and Bifidobacterium were opposite (P < 0.05). The Simpson index of the CRF group was lower than that of the control group, while the Chao index and Shannon index were opposite (P < 0.05). The levels of CD3+, CD4+, CD8+, and CD4+/CD8+ in the CRF group were lower than those in the control group, while the levels of CD14+, CD19+, and CD16+/CD56+ were opposite (P < 0.05). The intestinal flora Chao index of CRF group was negatively correlated with the levels of CD3+, CD4+, and CD8+ (r = −0.692, P=0.019; r = −0.669, P=0.021; r = −0.603, P=0.028). The intestinal flora Chao of CRF group is positively correlated with the level of CD14+ and CD16+/CD56+ (r = 0.615, P=0.026; r = 0.758, P=0.016). Conclusion There are intestinal flora disorder and the imbalance of immune function in CRF patients, which are mainly manifested in the change of intestinal flora structure, the increase of richness and diversity of intestinal flora, and the decrease of lymphocyte subgroups. There is correlation between the imbalance of intestinal colony and the imbalance of immune function.

Identification of miRNAs-mediated seed and stone-hardening regulatory networks and their signal pathway of GA-induced seedless berries in grapevine (V. vinifera L.)

BackgroundStone-hardening stage is crucial to the development of grape seed and berry quality. A significant body of evidence supports the important roles of MicroRNAs in grape-berry development, but their specific molecular functions during grape stone-hardening stage remain unclear.ResultsHere, a total of 161 conserved and 85 species-specific miRNAs/miRNAs* (precursor) were identified in grape berries at stone-hardening stage using Solexa sequencing. Amongst them, 30 VvmiRNAs were stone-hardening stage-specific, whereas 52 exhibited differential expression profiles during berry development, potentially participating in the modulation of berry development as verified by their expression patterns. GO and KEGG pathway analysis showed that 13 VvmiRNAs might be involved in the regulation of embryo development, another 11 in lignin and cellulose biosynthesis, and also 28 in the modulation of hormone signaling, sugar, and proline metabolism. Furthermore, the target genes for 4 novel VvmiRNAs related to berry development were validated using RNA Ligase-Mediated (RLM)-RACE and Poly(A) Polymerase-Mediated (PPM)-RACE methods, and their cleavage mainly occurred at the 9th–11th sites from the 5′ ends of miRNAs at their binding regions. In view of the regulatory roles of GA in seed embryo development and stone-hardening in grape, we investigated the expression modes of VvmiRNAs and their target genes during GA-induced grape seedless-berry development, and we validated that GA induced the expression of VvmiR31-3p and VvmiR8-5p to negatively regulate the expression levels of CAFFEOYL COENZYME A-3-O-METHYLTRANSFERASE (VvCCoAOMT), and DDB1-CUL4 ASSOCIATED FACTOR1 (VvDCAF1). The series of changes might repress grape stone hardening and embryo development, which might be a potential key molecular mechanism in GA-induced grape seedless-berry development. Finally, a schematic model of miRNA-mediated grape seed and stone-hardening development was proposed.ConclusionThis work identified 30 stone-hardening stage-specific VvmiRNAs and 52 significant differential expression ones, and preliminary interpreted the potential molecular mechanism of GA-induced grape parthenocarpy. GA negatively manipulate the expression of VvCCoAOMT and VvDCAF1 by up-regulation the expression of VvmiR31-3p and VvmiR8-5p, thereby repressing seed stone and embryo development to produce grape seedless berries.

MiR-125b regulates the differentiation of hair follicles in Fine-wool Sheep and Cashmere goats by targeting MXD4 and FGFR2

With the development of miRNAs identification technology, more and more miRNAs have been discovered, and the role of miRNAs in the development of animal hair follicles has become a focus of research on hair-producing animals. In the previous experiment, compare the microRNA (miRNA) trancriptomes of goats and sheep skin using Solexa sequencing and differentially expressed miR-125b was screened. However, the mechanism of miR-125b regulating hair follicle development is not clear. Therefore, in the present study, the expression of miR-125b, MXD4 and FGFR2 in skin tissue of Fine-wool Sheep and Cashmere goats and HEK-293T cells was examined by qPCR and Western blot. Furthermore, the correlation between miR-125b and the predicted target gene (MXD4, FGFR2) was verified using the Dual-luciferase Reporter assay. We demonstrated that the expression of MXD4 and FGFR2 in Cashmere goats was significantly higher than that of Fine-wool Sheep, and the expression was opposite to that of miR-125b. miR-125b can down-regulate the levels of MXD4 and FGFR2. Dual-luciferase reporter gene assay showed that miR-125b could bind to the 3′-UTR region of target genes FGFR2 and MXD4, suggesting that MXD4 and FGFR2 were target genes of miR-125b. This study has shown that the growth and development of hair follicles in skin tissue of Fine-wool Sheep and Cashmere goats from the new regulatory levels of miRNAs, and clarified the mechanism of miR-125b and its target genes in the development of hair follicles in the skin.

Transcriptome Profiling of Micromelalopha troglodyta (Lepidoptera: Notodontidae) Larvae under Tannin Stress Using Solexa Sequencing Technology

Abstract Tannins are a large group of polyphenolic compounds and natural protective substances for plant survival. The differentially expressed genes (DEGs) of Micromelalopha troglodyta (Graeser) under tannin stress were studied by Solexa sequencing technology. A total of 51,797,038–54,991,822 and 51,674,478–52,307,172 clean reads were obtained from the tannin treatment (TT) library and the control (CK) library transcriptomes, respectively, and assembled into 21,236 nonredundant consensus sequences. The expression of 1,627 unigenes in the TT library was remarkably different from that of the CK library; 885 genes were upregulated, and 742 genes were downregulated (P ≤ 0.001). The expression of 18 DEGs was detected by real-time fluorescent quantitative PCR, and the trend of gene expression was consistent with that of transcriptome data. In the biological process category, the DEGs were primarily related to cellular processes, metabolic processes, and single-organism processes. In the molecular function category, the DEGs were mainly involved in binding and catalytic activity, and in the cellular component category, the DEGs were mainly related to the cell, cell part, and organelle. Pathway enrichment analysis indicated that drug metabolism-cytochrome P450 and glutathione metabolism may be associated with detoxification-related processes under tannin stress, and glutathione S-transferases and other detoxification enzyme genes play an important role in detoxifying tannins in M. troglodyta larvae. This study also provides important resources for further study of the genes related to pesticide targets and metabolic processes in M. troglodyta.

Expression characteristics of pineal miRNAs at ovine different reproductive stages and the identification of miRNAs targeting the AANAT gene

BackgroundMany recent studies have shown that miRNAs play important roles in the regulation of animal reproduction, including seasonal reproduction. The pineal gland is a crucial hub in the regulation of seasonal reproduction. However, little is known about the expression characteristics of pineal miRNAs in different reproductive seasons (anestrus and breeding season). Therefore, the expression profiles and regulatory roles of ovine pineal miRNAs were investigated during different reproductive stages using Solexa sequencing technology and dual luciferase reporter assays.ResultsA total of 427 miRNAs were identified in the sheep pineal gland. Significant differences in miRNA expression were demonstrated between anestrus and the breeding season in terms of the frequency distributions of miRNA lengths, number of expressed miRNAs, and specifically and highly expressed miRNAs in each reproductive stage. KEGG analysis of the differentially expressed (DE) miRNAs between anestrus and the breeding season indicated that they are significantly enriched in pathways related to protein synthesis, secretion and uptake. Furthermore, transcriptome analysis revealed that many target genes of DE miRNAs in the ribosome pathway showed relatively low expression in the breeding season. On the other hand, analyses combining miRNA-gene expression data with target relationship validation in vitro implied that miR-89 may participate in the negative regulation of aralkylamine N-acetyltransferase (AANAT) mRNA expression by targeting its 3’UTR at a unique binding site.ConclusionsOur results provide new insights into the expression characteristics of sheep pineal miRNAs at different reproductive stages and into the negative regulatory effects of pineal miRNAs on AANAT mRNA expression.

PSXII-28 miRNA2382-5p regulates production of triglyceride in mammary epithelial cells via targeting NDRG2 of dairy cattle

Abstract Introduction: Based on the conjoint analysis of RNA-seq and Solexa sequencing of BMECs from dairy cattle with high and low milk fat percentages, miRNA2382-5p was screened as a differentially expressed miRNA, and NDRG2 might be the target gene of miRNA2382-5p. Materials and Methods: The experimental BMECs were previously constructed in our laboratory. The triglyceride Kit was used to separate the cells and detect the content of triglyceride. The target relationship of miRNA2382-5p and NDRG2 was validated by luciferase reporter assay, qRT-PCR and western blot. The data were analyzed by GraphPad 6.0. Results: The detection of triglyceride content in BMECs showed that the triglyceride in BMECs transfected with miRNA2382-5p mimics significantly decreased compared with BMECs transfected with control, and the triglyceride in BMECs transfected with miRNA2382-5p inhibitor significantly increased. The luciferase reporter assays revealed that the luciferase activity of BMECs transfected with pmiR-NDRG2-WT was decreased compared with BMECs transfected with pmiRR-NDRG2-mut and pmiR-REPORT. The qRT-PCR and western blot result showed that the expression of NDRG2 in BMECs transfected with miRNA2382-5p mimics was significantly decreased, and the expression of NDRG2 in BMECs transfected with the miRNA2382-5p inhibitor was significantly increased compared with BMECs transfected with control. Moreover, compared with control group, the triglyceride in BMECs transfected with pBI-CMV3-NDRG2 was significantly increased, and the pGPU6/GFP/Neo-NDRG2 group was significantly decreased. Discussion and Conclusions: Comparative proteomics analysis of longissimus muscle from different breeds of cattle showed that NDRG2 was positively correlated with intramuscular fat deposition. In our study, we screened NDRG2 as a DEG in both the RNA-seq and conjoint analyses and confirmed that NDRG2 is a target gene of miRNA2382-5p. MiRNA2382-5p can inhibit the production of triglyceride, and the overexpression of NDRG2 can promote the production of triglyceride. Therefore, we believe that miRNA2382-5p can affect triglyceride production by negatively regulating the expression of NDRG2.

Expression of miRNAs and their target genes in roots of ‘Sanhu’ tangerine (Citrus reticulata blanco cv. ‘Sanhu’) in response to Candidatus Liberibacter asiaticus infection

MicroRNA (miRNA) profiles were studied in roots of ‘Sanhu’ tangerine plants with and without Candidatus Liberibacter asiaticus (CLas) infections by using solexa sequencing to investigate the possible involvement of miRNAs in the pathogenesis of Citrus Greening (Huanglongbing) disease. A total of 186 known miRNAs and 71 novel miRNAs were thus identified. Of them, 37 known and 18 novel miRNAs were significantly different in their expression patterns in roots of CLas-free and CLas-infected plants. CLas infection induced de novo expression of 6 known miRNAs and up-regulated the expression of 13 known miRNAs. It also prevented the expression of 11 known miRNAs and down-regulated the expression of 7 others. A total of 316 genes were predicted to be the targets of these differentially expressed miRNAs. Particularly, 34, 28, 21, and 20 genes were predicted to be the respective targets of cre-miR156a, cre-miR396b, cre-miR396g-5p, and cre-miRn70. The predicted putative target genes are known to be involved in stress response, plant growth and development, transcription, and metabolism. Quantitative analyses of the expression of several representative miRNAs and their target genes were generally in agreement with the predicted expression patterns (inverse proportion). Further research to unravel the interactions between these miRNAs and their target genes and determine the associated biological and pathological significance is recommended.

Screening for Candidate Genes Associated with Biocontrol Mechanisms of Bacillus pumilus DX01 Using Tn5 Transposon Mutagenesis and a 2-DE-Based Comparative Proteomic Analysis.

A total of 1467 mutants of the biocontrol bacterium Bacillus pumilus DX01 were obtained by Tn5 insertional mutagenesis and subjected to the determination of antagonistic capabilities. Compared with the wild-type strain DX01, the mutant M25 was identified to have the most significant reduction in antagonistic capability against the phytopathogen Bipolaris maydis and extracellular proteinase activity. The integration site of the exogenous T-DNA in the genome of mutant M25 was revealed in the coding region of malony CoA-ACP transacylase (MCAT) gene (mcat), which belongs to a polyketide synthase (PKS) gene cluster, DX01pks of B. pumilus DX01. Furthermore, the whole DX01pks gene cluster was cloned using Illumina Solexa sequencing technology, and it has a modular framework different from the other two gene clusters involved in polyketide synthesis in B. amyloliquefaciens FZB42 (pks1) and B. subtilis 168 (pksX). Finally, in order to gain more insights into the molecular mechanisms of biocontrol of B. pumilus DX01, the changes in the relative level of expression of total proteins between the original strain DX01 and the mutant M25 were detected by 2-DE-based proteomic analysis. A total of twenty differentially expressed proteins were identified upon the mcat gene transposition mutagenesis. Of these proteins, seven proteins were up-regulated in expression level and the other proteins were down-regulated. Taken together, the results in this study showed that Tn5 transposon mutagenesis of B. pumilus DX01 can lead to a significant change of antiphytopathogen ability, and the DX01pks gene cluster possibly play a potential role in the biocontrol processes of this bacterium.

Study on potential biomarkers of energy metabolism-related to early-stage Yin-deficiency-heat syndrome based on metabolomics and transcriptomics.

Yin-deficiency-heat (YDH) syndrome is a common sub-health state of the human body in traditional Chinese medicine (TCM). However, due to the lack of objective quantitative diagnostic indicators, patients with early-stage YDH syndrome cannot be treated in time and can develop a pathological (disease) state. Therefore, it is necessary to apply modern diagnostic techniques in order to identify the biological markers for the diagnosis of early-stage YDH syndrome. In the present study, we performed Solexa sequencing and non-targeted metabolomics analysis using high-performance liquid chromatography coupled with mass spectrometry to screen differentially expressed mRNAs and differential metabolites in individuals with early-stage YDH syndrome and healthy controls. Bioinformatics methods were used to perform enrichment analysis of differentially expressed mRNAs and differential metabolites for biological functions and signaling pathways. Furthermore, we found that differentially expressed mRNAs and differential metabolites were related to energy metabolism. Real-time PCR was used to validate the mRNA expression in the serum of subjects with early-stage YDH syndrome. We found that the mitochondrially encoded NADH dehydrogenase 2 (MT-ND2) mRNA was differentially expressed in the serum of individuals with early-stage YDH syndrome. Receiver operating characteristic (ROC) curve and logistic regression analysis were used to evaluate the efficacy of the diagnostic model based on eight differential metabolites. We combined the three metabolites such as Glycine, Sphingomyelin, and Isocitrate to establish the diagnostic model with a sensitivity of 0.853 and a specificity of 0.800. The combination of the above three metabolites may serve as a potential biomarker for the diagnosis of early-stage YDH syndrome. Our study reveals potential biomarker for the diagnosis of early-stage YDH syndrome and also provides a new method for the quantification and objectification of TCM syndromes.

Different Diets Change the Expression of Bovine Serum Extracellular Vesicle-miRNAs.

Simple SummaryStudies over the last decade have shown that cells can communicate with neighboring or distant cells through complex packets stuffed with selected proteins, lipids, and nucleic acids, called extracellular vesicles. The wrapped macromolecules are miRNAs, which play a central role in mediating the signal communication of creatural patho/physiological systems. Extracellular vesicle-miRNAs vary among species and different body fluids, such as milk, urine, saliva, cerebrospinal fluid and blood, providing general and individual characters of the vesicles. Cow’s milk is significant in the supply of human nutrition. Therefore, the extracellular vesicle-related physiological process of dairy cows should be of concern. This study clarified the miRNA profiling of bovine serum and found their potential influence on immunity. Moreover, we found that different diets could affect miRNA expression. The results implied that people could implement effective dietary strategies to intervene in the physiological state of animals.Cells can communicate with neighboring or distant cells using extracellular vesicles (EVs), mainly attributed to their containing miRNAs. Given that diets can change host circulatory miRNA profiling, and EVs are the major miRNA carriers in serum, we hypothesized that different diets could change bovine circulating EV-miRNA expression. We partly replaced alfalfa hay with whole cotton seed and soybean hull in the feed formula of the tested cows. Blood EVs were isolated using a polyethylene glycol precipitation kit. Particle size analysis revealed exosomes were dominant in bovine serum EVs. Small RNAs were enriched in bovine serum EVs, including miRNAs, snRNAs, tiRNAs, Cis-regulatory elements, piRNAs, etc. In total, 359 types of Bos taurus miRNAs were identified by Solexa sequencing. Each cow in the control group contained about 244 types of serum EV-miRNAs, compared to 246 types in the tested group. There were 15 immune-related miRNAs in the top 20 serum EV-miRNAs, accounting for about 80% of the total. Seven differently expressed known miRNAs were detected in responding to different diets. An analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed differently expressed miRNAs were related to hormone signal pathways and protein metabolism. Bovine serum EVs are abundant with miRNAs, most of which are immune-related. Different diets eventually change the miRNA profiling of bovine serum EVs.

Analysis of miRNAs in the Heads of Different Castes of the Bumblebee Bombus lantschouensis (Hymenoptera: Apidae).

Bumblebees are important insect pollinators for many wildflowers and crops. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that regulate different biological functions in insects. In this study, the miRNAs in the heads of the three castes of the bumblebee Bombus lantschouensis were identified and characterized by small RNA deep sequencing. The significant differences in the expression of miRNAs and their target genes were analyzed. The results showed that the length of the small RNA reads from males, queens, and workers was distributed between 18 and 30 nt, with a peak at 22 nt. A total of 364 known and 89 novel miRNAs were identified from the heads of the three castes. The eight miRNAs with the highest expressed levels in males, queens, and workers were identical, although the order of these miRNAs based on expression differed. The male vs. queen, male vs. worker, and worker vs. queen comparisons identified nine, fourteen, and four miRNAs with significant differences in expression, respectively. The different castes were clustered based on the differentially expressed miRNAs (DE miRNAs), and the expression levels of the DE miRNAs obtained by RT-qPCR were consistent with the read counts obtained through Solexa sequencing. The putative target genes of these DE miRNAs were enriched in 29 Gene Ontology (GO) terms, and catalytic activity was the most enriched GO term, as demonstrated by its association with 2837 target genes in the male vs. queen comparison, 3535 target genes in the male vs. worker comparison, and 2185 target genes in the worker vs. queen comparison. This study highlights the characteristics of the miRNAs in the three B. lantschouensis castes and will aid further studies on the functions of miRNAs in bumblebees.

Identification and characterization of microRNAs in the gonad of Trachinotus ovatus using Solexa sequencing

The Trachinotus ovatus (T. ovatus) reach sexual maturity as late as 4–5 years, and there are no obvious morphology differences between males and females, even at maturity, making the selection of male and female parents for selective breeding difficult. To examine the potential regulatory mechanism of sexual differentiation, we conducted a microRNAs (miRNAs) analysis on the ovaries and testes of T. ovatus. A total of 13,423,691 and 11,734,953 raw reads, representing 91,883,908 and 86,879,726 unique sequences of 18–35 nt length obtained from the ovaries and testes, respectively. After mapping to the T. ovatus transcriptome reference sequence (unpublished data), and comparing the miRNA sequences with the miRBase database, 179 known mature miRNAs and 100 novel miRNAs were identified. Expression analysis showed that, 165 miRNAs were differentially expressed between the ovary and testis.To validate the Illumina results, the expression patterns of the nine most differently expressed miRNAs (dre-miR-7a, dre-miR-7b, dre-miR-153a-3p, dre-miR-144-3p, dre-miR-301a, dre-miR-92a-3p, dre-miR-727-5p, Novel 124, and Novel 190, as well as those of five miRNAs that may relate to gonad development (dre-miR-143, dre-miR-101a, dre-miR-202-5p, dre-let-7c-5p, and dre-miR-181a-5p), were assayed with RT-qPCR. The expression trends for all the miRNAs validated with RT-qPCR were consistent with the next-generation sequencing data. The identification and characterization of the miRNAs differentially expressed between the ovary and the testis of the T. ovatus will increase our understanding of the role of miRNAs in gonad differentiation. These data also facilitate studies on miRNA regulation of teleost reproduction.

HBV-Encoded miR-2 Functions as an Oncogene by Downregulating TRIM35 But Upregulating RAN in Liver Cancer Cells

Hepatitis B virus (HBV) infection has been well established as a high-risk factor for the carcinogenesis of hepatocellular carcinoma (HCC). Cellular microRNA (miRNA) is involved in tumorigenesis by accelerating the malignant phenotype in HCC. However, whether HBV can encode miRNAs that contribute to HCC is not entirely clear. In this study, an miRNA encoded by HBV (HBV-miR-2) was identified by Solexa sequencing in HBV-positive HCC specimens and further verified in serum samples from HCC patients with HBV infection and in HBV-positive HCC cell lines. We revealed that HBV-miR-2 promoted HCC cell growth ability by suppressing apoptosis and promoting migration and invasion by enhancing the epithelial-mesenchymal transition (EMT), functioning as an oncogene in the development of HBV-related HCC. Furthermore, we demonstrated that HBV-miR-2 suppresses the expression of TRIM35 but enhances RAN expression by targeting their 3'-untranslated regions (3'UTR) and that the ectopic expression of TRIM35 or knockdown of RAN counteracted the malignant phenotypes induced by HBV-miR-2. Funding Statement: This work was supported in part by the National Natural Science Foundation of China (No: 81830094; 91629302; 81572790; 31270818) and the Natural Science Foundation of Tianjin (No: 12JCZDJC25100). Declaration of Interests: All authors declare that there are no potential conflicts of interest. Ethics Approval Statement: The study was approved by the ethics committee of Tianjin Medical University.

De novo transcriptomic analysis of gonad of Strongylocentrotus nudus and gene discovery for biosynthesis of polyunsaturated fatty acids.

Strongylocentrotus nudus is an important cultured sea urchin species in north China, because its gonad is rich in unsaturated fatty acids, particularly long polyunsaturated fatty acids (LC-PUFAs). These PUFAs play pleiotropic and crucial roles in a wide range of biological process. However, the genes contributing to biosynthesis PUFAs have not been elucidated yet, and the molecular mechanism relative to the difference in PUFA composition between male and female gonad as been revealed but the corresponding has not been understood. In this paper, solexa sequencing based transcriptomic approach was used to identify and characterize the key genes relative to PUFA synthesis and further conducted different expressed genes between male and female gonad. A total of 130,124 transcripts and 189330 unigenes were de novo assembled from 64.32Gb data. Next, these unigenes were subjected to functional annotation by mapping to six public databases, and this process revealed a lot of genes involving in lipid metabolism. In addition, three types of fatty acids front-end desaturase and three species of very long fatty acids elongase were identified and the pathway for PUFA biosynthesis was hypothesized. Last, comparative analysis revealed the higher expression level of Δ5 desaturase, Δ6 desaturase, ELOVL-4, -6 and -7 in male gonad compared with female. This results could plausible explain the differ in composition of PUFAs between male and female gonad of sea urchin.

Upregulated miR-154 promotes ECM degradation in intervertebral disc degeneration.

Intervertebral disc degeneration (IDD), a common global health issue, is a major cause for low back pain (LBP). Given the complex etiology of IDD, micro RNA (miRNA) recently has been demonstrated to play essential roles in the progression of IDD. Therefore, this study aims to investigate functions of the miR-154, which is well-documented in a series of cell activities, IDD, and other relevant mechanisms. Lumbar nucleus pulposus (NP) samples were collected from patients with IDD and the control group. After solexa sequencing and bioinformatical analysis, the results showed that miR-154 was increased in NP cells of patients with IDD. Inhibition of miR-154 increased type II collagen and aggrecan and decreased mRNA expressions of collagenase-3 (MMP13) and aggrecanase-1 (ADAMTS4), whereas overexpression of miR-154 reversed such effects in NP cells. In addition, the luciferase reporter assay revealed that fibroblast growth factor 14 (FGF14) is a direct target of miR-154 and that the overexpression of FGF14 leads to similar effects as inhibition of miR-154 did. In conclusion, the results suggested that miR-154 participates in the development of IDD and its effects are mediated via targeting FGF14. Thus, miR-154 may be thought as a potential etiological factor for IDD and may provide insights into a therapeutic target to treat IDD.