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Background: Tan and Hu sheep are well-known local breeds in China, producing lamb fur with unique ornamental and practical values highly appreciated by consumers worldwide. Fur quality is optimal at one month of age and gradually declines with time. Despite active research on its genetic mechanism using transcriptomic and whole genome bisulfite sequencing analysis, the main effective gene locus has not been found, and its regulatory mechanism is still unclear, which limits the breeding and improvement of fur traits. Results: Scapular skin samples from newborn (1-month old) and adult (24-month old) Tan sheep were utilized for small ribonucleic acid (RNA) sequencing Principal Component Analysis (PCA) showed that the newborn and adult groups were completely separated. Differential expression analysis of micro-RNAs (miRNAs) identified 32 up-regulated miRNAs and 48 down-regulated miRNAs in the newborn groups. All up-regulated miRNAs were located in the imprinted. Dlk1-Gtl2 locus on chromosome 18, whereas all down-regulated miRNAs were distributed across the sheep chromosomes, without a clear pattern of positional consistency. Further, by systematically analyzing the target genes and signaling pathways of all 32 up-regulated miRNAs, we found that the PI3K-AKT signaling pathway has the potential to be targeted and regulated by most of the miRNAs in the Dlk1-Gtl2 region. In addition, we also re-analyzed miRNA sequencing data from public databases on Hu lambs (full sibling Hu lambs with high- and low-quality fur characteristics). Again, it was found that most of the up-regulated miRNAs in lambs with high-quality fur were also located in the Dlk1-Gtl2 region, whereas this patter was not present for down-regulated miRNAs. Conclusion: Sequencing of miRNAs in conjunction with public databases was employed to identify miRNAs within the imprinted Dlk1-Gtl2 region on chromosome 18, suggesting their potential roles as epigenetic regulators of fur traits. Small RNAs located at the Dlk1-Gtl2 locus were identified as having the potential to systematically regulate the PI3K-AKT signaling pathway, thereby indicating the relevance of the Dlk1-Gtl2/PI3K-AKT axis in the context of fur traits. Selection of parental specific expressed imprinted genes in the process of conserving and exploiting lamb fur traits should be emphasized. [ABSTRACT FROM AUTHOR]

Background: Macrophages play an important role in regulating the course of spinal tuberculosis within the bone microenvironment. This study aimed to investigate the differential expression of miRNA in macrophage-derived exosomes within the tuberculosis-infected bone microenvironment, to identify miRNAs that hold potential as diagnostic markers and therapeutic targets. Methods: We established study cohorts for spinal tuberculosis, collected bone marrow blood samples, isolated macrophage exosomes, and performed exosome miRNA sequencing. A miRNA-mRNA co-expression network was constructed using WGCNA analysis. Gene GO analysis and KEGG pathway enrichment analysis were performed using KOBAS software. Target miRNAs were selected based on fold change, P-value, and false discovery rate, and their validation was carried out using qRT-PCR and ROC curve studies. Subsequently, we constructed a target gene network for these miRNAs and performed KEGG pathway enrichment analysis to explore the potential signaling mechanisms involved in regulating the disease course of spinal tuberculosis. Results: Our findings revealed that macrophages from the tuberculosis-infected bone microenvironment exhibited an M1 phenotype. The successful extraction of exosomes from macrophage supernatants was confirmed through electron microscopy, particle size analysis, and protein blot analysis. Exosome miRNAseq demonstrated that 28 miRNAs were up-regulated, while 34 miRNAs were down-regulated in individuals with spinal tuberculosis. GO analysis and KEGG pathway enrichment analysis indicated that the differentially expressed miRNAs were involved in various biological processes, cell components, molecular functions, and signaling pathways, which collectively contribute to the regulation of the disease course of spinal tuberculosis. Notably, miRNA-125b-5p was successfully selected based on fold change, p-value, and false discovery rate. qRT-PCR validation further confirmed the significant up-regulation of miRNA-125b-5p in spinal tuberculosis. The ROC curve revealed that miR125b-5p is a potential diagnostic biomarker for spinal tuberculosis. Moreover, construction of the miRNA-125b-5p target gene network and subsequent KEGG enrichment analysis highlighted the importance of MAPK, TNF, Ras, Rap1, and the PI3K-Akt signaling pathways in the regulation of the disease course of spinal tuberculosis. Conclusion: Our study demonstrates differential expression of miRNAs in macrophage-derived exosomes in the tuberculosis-infected bone microenvironment. Specifically, MiRNA-125b-5p is significantly up-regulated in spinal tuberculosis and shows potential as a diagnostic biomarker for spinal tuberculosis. [ABSTRACT FROM AUTHOR]

MicroRNA transcriptomes from fresh tumors and the adjacent normal tissues were profiled in 10 Korean patients diagnosed with lung adenocarcinoma using a next-generation sequencing (NGS) technique called miRNA-seq. The sequencing quality was assessed using FastQC, and low-quality or adapter-contaminated portions of the reads were removed using Trim Galore. Quality-assured reads were analyzed using miRDeep2 and Bowtie. The abundance of known miRNAs was estimated using the reads per million (RPM) normalization method. Subsequently, using DESeq2 and Wx, we identified differentially expressed miRNAs and potential miRNA biomarkers for lung adenocarcinoma tissues compared to adjacent normal tissues, respectively. We defined reliable miRNA biomarkers for lung adenocarcinoma as those detected by both methods. The miRNA-seq data are available in the Gene Expression Omnibus (GEO) database under accession number GSE196633, and all processed data can be accessed via the Mendeley data website. Dataset:  https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE196633 and https://data.mendeley.com/datasets/vp977psjcb/2. Dataset License: CC0 [ABSTRACT FROM AUTHOR]

Exosomes cargo tumour-characterized biomolecules secreted from cancer cells and play a pivotal role in tumorigenesis and cancer progression, thus providing their potential for non-invasive cancer monitoring. Since cancer cell-derived exosomes are often mixed with those from healthy cells in liquid biopsy of tumour patients, accurately measuring the purity of tumour cell-derived exosomes is not only critical for the early detection but also essential for unbiased identification of diagnosis biomarkers. Here, we propose 'ExosomePurity', a tumour purity deconvolution model to estimate tumour purity in serum exosomes of cancer patients based on microribonucleic acid (miRNA)-Seq data. We first identify the differently expressed miRNAs as signature to distinguish cancer cell- from healthy cell-derived exosomes. Then, the deconvolution model was developed to estimate the proportions of cancer exosomes and normal exosomes in serum. The purity predicted by the model shows high correlation with actual purity in simulated data and actual data. Moreover, the model is robust under the different levels of noise background. The tumour purity was also used to correct differential expressed gene analysis. ExosomePurity empowers the research community to study non-invasive early diagnosis and to track cancer progression in cancers more efficiently. It is implemented in R and is freely available from GitHub (https://github.com/WangHYLab/ExosomePurity). [ABSTRACT FROM AUTHOR]

Myometrial contraction is essential for successful delivery. Recent studies have highlighted the vital roles of tissue-derived exosomes in disease diagnostic, prognostic, and therapeutic applications; however, the characteristics of uterine myometrium-derived exosomes are unclear. Here, we successfully isolated exosomes from myometrial tissues, human myometrial smooth muscle cells (HMSMCs), and human umbilical vein endothelial cells (HUVECs), then performed quantitative liquid chromatography-tandem mass spectrometry and miRNA sequencing to investigate the cargo of the exosomes. Fifty-two proteins and five miRNAs were differentially expressed (DE) in term non-labor and term labor myometrium-derived exosomes. Among them, seven proteins (SERPINE1, THBS1, MGAT1, VIM, FGB, FGG, and VWF) were differentially expressed both in the myometrial exosomes and tissues, three miRNAs (miR-363-3p, miR-203a-3p, and miR-205-5p) target 13 DE genes. The top three miRNA derived from HMSMCs (miR-125b-1-3p, miR-337-5p, and miR-503-5p) and HUVECs (miR-663a, miR-4463, and miR-3622a-5p) were identified. Two proteins, GJA1 and SLC39A14, exist in female blood exosomes and are highly expressed in HMSMCs exosomes, are also upregulated in the laboring myometrium, which verified increased in laboring blood samples, might be novel potential biomarkers for myometrial activation. The proteomic and miRNA profile of exosomes derived from laboring myometrium revealed some molecules in the exosomes that affect the intercellular communication and the function of the myometrium. [ABSTRACT FROM AUTHOR]

Pre-analytical factors have a significant influence on circulating microRNA (miRNA) profiling. The aim of this study was a comprehensive assessment of the impact of the anticoagulant type in blood collection tubes on circulating plasma miRNA profiles using small RNA sequencing. Blood from ten healthy participants (five males and five females from 25 to 40 years old) was taken in collection tubes with four different anticoagulants: acid citrate dextrose (ACD-B), sodium citrate, citrate-theophylline-adenosine-dipyridamole (CTAD) and dipotassium-ethylenediaminetetraacetic acid (K2 EDTA). Platelet-free plasma samples were obtained by double centrifugation. EDTA plasma samples had elevated levels of hemolysis compared to samples obtained using other anticoagulants. Small RNA was extracted from plasma samples and small RNA sequencing was performed on the Illumina NextSeq 500 system. A total of 30 samples had been successfully sequenced starting from ~1 M reads mapped to miRNAs, allowing us to analyze their diversity and isoform content. The principal component analysis showed that the EDTA samples have distinct circulating plasma miRNA profiles compared to samples obtained using other anticoagulants. We selected 50 miRNA species that were differentially expressed between the sample groups based on the type of anticoagulant. We found that the EDTA samples had elevated levels of miRNAs which are abundant in red blood cells (RBC) and associated with hemolysis, while the levels of some platelet-specific miRNAs in these samples were lowered. The ratio between RBC-derived and platelet-derived miRNAs differed between the EDTA samples and other sample groups, which was validated by quantitative PCR. This study provides full plasma miRNA profiles of 10 healthy adults, compares them with previous studies and shows that the profile of circulating miRNAs in the EDTA plasma samples is altered primarily due to an increased level of hemolysis. [ABSTRACT FROM AUTHOR]

We aimed to identify miRNAs and pathways specifically deregulated in adolescent and young adult (AYA) T-ALL patients. Small RNA-seq showed no major differences between AYA and pediatric T-ALL, but it revealed downregulation of miR-143-3p in T-ALL patients. Prediction algorithms identified several known and putative oncogenes targeted by this miRNA, including KRAS, FGF1, and FGF9. Pathway analysis indicated signaling pathways related to cell growth and proliferation, including FGFR signaling and PI3K-AKT signaling, with the majority of genes overrepresented in these pathways being predicted targets of hsa-miR-143-3p. By luciferase reporter assays, we validated direct interactions of this miRNA with KRAS, FGF1 and FGF9. In cell proliferation assays, we showed reduction of cell growth upon miR-143-3p overexpression in two T-ALL cell lines. Our study is the first description of the miRNA transcriptome in AYA T-ALL patients and the first report on tumor suppressor potential of miR-143-3p in T-ALL. Downregulation of this miRNA in T-ALL patients might contribute to enhanced growth and viability of leukemic cells. We also discuss the potential role of miR-143-3p in FGFR signaling. Although this requires more extensive validation, it might be an interesting direction, since FGFR inhibition proved promising in preclinical studies in various cancers. [ABSTRACT FROM AUTHOR]

MicroRNA transcriptomes from fresh tumors and the adjacent normal tissues were profiled in 10 Korean patients diagnosed with lung adenocarcinoma using a next-generation sequencing (NGS) technique called miRNA-seq. The sequencing quality was assessed using FastQC, and low-quality or adapter-contaminated portions of the reads were removed using Trim Galore. Quality-assured reads were analyzed using miRDeep2 and Bowtie. The abundance of known miRNAs was estimated using the reads per million (RPM) normalization method. Subsequently, using DESeq2 and Wx, we identified differentially expressed miRNAs and potential miRNA biomarkers for lung adenocarcinoma tissues compared to adjacent normal tissues, respectively. We defined reliable miRNA biomarkers for lung adenocarcinoma as those detected by both methods. The miRNA-seq data are available in the Gene Expression Omnibus (GEO) database under accession number GSE196633, and all processed data can be accessed via the Mendeley data website.

Purpose: Hypoxia plays an essential role in the progression of hepatocellular carcinoma (HCC), whereas hypoxia inducible factor-1 (HIF-1) is the key transcription factor allowing HCC to survive hypoxia. The aim of this study was to define the essential mRNAs and miRNAs regulated by HIF1A and dissect their functions, interactions, and tumor-infiltrating immune cells in HCC. Methods: A human HCC cell line HepG2 was used as a cell model of HCC. The CRISPR/Cas9 system was used to knock out HIF1A in HepG2 cells, and RNA sequencing was utilized to characterize differentially expressed mRNAs and miRNAs in the HIF1A -knockout HepG2 cells; the identified candidates were then analyzed by GO annotation and KEGG pathway enrichment to study their function and establish a PPI network. Quantitative (q) PCR was used to verify if there were significant differences in the expression of mRNAs, and the association of the selected mRNAs expression with immune cell infiltration levels was further analyzed using The Cancer Genome Atlas (TCGA) pan-cancer data. Results: Using RNA-sequencing, we discovered that there were 1535 mRNAs differentially expressed (adjusted p < 0.05, |fold change|>1.5) in the HIF1A -knockout HepG2 cells, among which there were 644 mRNAs upregulated and 891 mRNAs downregulated. GO annotation and KEGG pathway enrichment showed that these mRNAs were involved in glycolysis/gluconeogenesis, PI3K-Akt signaling pathways, and HIF-1 signaling pathways. In addition, we found that there were 309 miRNAs differentially expressed (adjusted p < 0.05, |fold change|>1.5) in the HIF1A-knockout HepG2 cells, of which there were 213 miRNAs upregulated and 96 miRNAs downregulated. Our further analyses uncovered that these miRNA putative targets were involved in the hippo signaling pathway, axon guidance, and tight junction. Moreover, the construction and analysis of the PPI network showed that OASL , IL6 , and TAF1 were recognized as hub genes with the highest connectivity degrees. Importantly, in the HIF1A -knockout HepG2 cells, our qRT-PCR data confirmed the selected mRNA changes revealed by RNA-sequencing, and with TCGA pan-cancer data, we revealed that the expressional levels of these three genes, LUM , SCOC , and CCL2 , were associated with immune cell infiltration levels. Conclusion: The identified potential key network of mRNAs and miRNAs regulated by HIF1A in the HCC cells suggests a key role of HIF1A in the tumorigenesis of HCC. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder, with mutations in hundreds of genes contributing to its risk. Herein, we studied lymphoblastoid cell lines (LCLs) from children diagnosed with autistic disorder (n = 10) and controls (n = 7) using RNA and miRNA sequencing profiles. The sequencing analysis identified 1700 genes and 102 miRNAs differentially expressed between the ASD and control LCLs (p ≤ 0.05). The top upregulated genes were GABRA4, AUTS2, and IL27, and the top upregulated miRNAs were hsa-miR-6813-3p, hsa-miR-221-5p, and hsa-miR-21-5p. The RT-qPCR analysis confirmed the sequencing results for randomly selected candidates: AUTS2, FMR1, PTEN, hsa-miR-15a-5p, hsa-miR-92a-3p, and hsa-miR-125b-5p. The functional enrichment analysis showed pathways involved in ASD control proliferation of neuronal cells, cell death of immune cells, epilepsy or neurodevelopmental disorders, WNT and PTEN signaling, apoptosis, and cancer. The integration of mRNA and miRNA sequencing profiles by miRWalk2.0 identified correlated changes in miRNAs and their targets' expression. The integration analysis found significantly dysregulated miRNA–gene pairs in ASD. Overall, these findings suggest that mRNA and miRNA expression profiles in ASD are greatly altered in LCLs and reveal numerous miRNA–gene interactions that regulate critical pathways involved in the proliferation of neuronal cells, cell death of immune cells, and neuronal development. [ABSTRACT FROM AUTHOR]

Infection with Salmonella Typhimurium (S. Typhimurium) is a common cause of food-borne zoonosis leading to acute gastroenteritis in humans and pigs, causing economic losses to producers and farmers, and generating a food security risk. In a previous study, we demonstrated that S. Typhimurium infection produces a severe transcriptional activation of inflammatory processes in ileum. However, little is known regarding how microRNAs regulate this response during infection. Here, small RNA sequencing was used to identify 28 miRNAs differentially expressed (DE) in ileum of S. Typhimurium-infected pigs, which potentially regulate 14 target genes involved in immune system processes such as regulation of cytokine production, monocyte chemotaxis, or cellular response to interferon gamma. Using in vitro functional and gain/loss of function (mimics/CRISPR-Cas system) approaches, we show that porcine miR-194a-5p (homologous to human miR-194-5p) regulates TLR4 gene expression, an important molecule involved in pathogen virulence, recognition and activation of innate immunity in Salmonella infection. [ABSTRACT FROM AUTHOR]

Throughout the world, numerous individuals are infected with Toxoplasma gondii, which may improve immunity against cancer. Furthermore, microRNAs (miRs) may be differentially expressed in the host upon infection with T. gondii. In the present study, RNA-sequencing analysis and reverse transcription-quantitative PCR revealed that miR-429-3p, miR-145a-5p, miR-211-5p, miR-31-3p and miR-135a-5p were determined to be downregulated, while miR-21a-3p, miR-135b-5p, miR-210-5p and miR-146-3p were upregulated in mice post-infection with T. gondii. Antitumor genes [TNF receptor superfamily member 11b, large tumor suppressor kinase (Lats)2 and Lats1] were identified as targets of miR-429-3p, miR-145a-5p, miR-211-5p, miR-31-3p and miR-135a-5p with a luciferase reporter assay. In addition, the protein levels of Lats2 and Lats1 were detected to be higher in T. gondii-infected mice than in the control group. Therefore, these results provide favorable evidence for the suppression of cancer upon T. gondii infection and may give novel ideas for the treatment of tumors. [ABSTRACT FROM AUTHOR]

By analyzing the sequencing results, we found that PKM2 was significantly enriched in the KEGG pathway associated with muscle development and was screened as a hub gene in the fast-growing group; hence, we speculate that PKM2 may be associated with the growth and development of Bian chickens. Keywords: miRNA-seq; skeletal muscle; Bian chicken; growth and development EN miRNA-seq skeletal muscle Bian chicken growth and development N.PAG N.PAG 15 04/26/22 20220415 NES 220415 1. Apart from research on genes related to Bian chicken growth, there are few studies on miRNA functions in Bian chicken growth. miRNA is a noncoding sequence with a length of approximately 22 nt, which inhibits the expression of target genes by binding to the 3'UTR region of mRNA [[5]]. In order to further explore the key DEMs and predicted target genes, we performed an interaction network analysis of genes enriched in KEGG pathways associated with muscle growth and development. [Extracted from the article]

Tetraploid Robinia pseudoacacia L. is a difficult-to-root species, and is vegetatively propagated through stem cuttings. Limited information is available regarding the adventitious root (AR) formation of dark-pretreated micro-shoot cuttings. Moreover, the role of specific miRNAs and their targeted genes during dark-pretreated AR formation under in vitro conditions has never been revealed. The dark pretreatment has successfully promoted and stimulated adventitious rooting signaling-related genes in tissue-cultured stem cuttings with the application of auxin (0.2 mg L−1 IBA). Histological analysis was performed for AR formation at 0, 12, 36, 48, and 72 h after excision (HAE) of the cuttings. The first histological events were observed at 36 HAE in the dark-pretreated cuttings; however, no cellular activities were observed in the control cuttings. In addition, the present study aimed to uncover the role of differentially expressed (DE) microRNAs (miRNAs) and their targeted genes during adventitious root formation using the lower portion (1–1.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points. Seven DE miRNA libraries were constructed and sequenced. The DE number of 81, 162, 153, 154, 41, 9, and 77 miRNAs were upregulated, whereas 67, 98, 84, 116, 19, 16, and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72, respectively. Furthermore, we depicted an association between ten miRNAs (novel-m0778-3p, miR6135e.2-5p, miR477-3p, miR4416c-5p, miR946d, miR398b, miR389a-3p, novel m0068-5p, novel-m0650-3p, and novel-m0560-3p) and important target genes (auxin response factor-3, gretchen hagen-9, scarecrow-like-1, squamosa promoter-binding protein-like-12, small auxin upregulated RNA-70, binding protein-9, vacuolar invertase-1, starch synthase-3, sucrose synthase-3, probable starch synthase-3, cell wall invertase-4, and trehalose phosphatase synthase-5), all of which play a role in plant hormone signaling and starch and sucrose metabolism pathways. The quantitative polymerase chain reaction (qRT-PCR) was used to validate the relative expression of these miRNAs and their targeted genes. These results provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in woody plants. [ABSTRACT FROM AUTHOR]

Pasteurella multocida (P. multocida) is a zoonotic bacterium that predominantly colonizes the respiratory tract and lungs of a variety of farmed and wild animals, and causes severe respiratory disease. To investigate the characteristics of the host immune response induced by P. multocida strains of serotype A and D, high-throughput mRNA-Seq and miRNA-Seq were performed to analyze the changes in goat bronchial epithelial cells stimulated by these two serotypes of P. multocida for 4 h. Quantitative RT-PCR was used to validate the randomly selected genes and miRNAs. The results revealed 204 and 117 differentially expressed mRNAs (jlog2(Fold-change)j - 1, p-value < 0.05) in the P. multocida serotype A and D stimulated groups, respectively. Meanwhile, the number of differentially expressed miRNAs (jlog2(Fold-change)j>0:1, p-value < 0.05) were 269 and 290, respectively. GO and KEGG enrichment analyses revealed 13 GO terms (p-value < 0.05) and four KEGG pathways (p-value < 0.05) associated with immunity. In the serotype A-stimulated group, the immune-related pathways were the GABAergic synapse and Toll-like receptor signaling pathways, while in the serotype D-stimulated group, the immune-related pathways were the phagosome and B cell receptor signaling pathways. Based on the predicted results of TargetScan and miRanda, the differentially expressed mRNA-miRNA network of immune-related GO terms and KEGG pathways was constructed. According to the cell morphological changes and the significant immune-related KEGG pathways, it was speculated that the P. multocida serotype D strain-stimulated goat bronchial epithelial cells may induce a cellular immune response earlier than serotype A-stimulated cells. Our study provides valuable insight into the host immune response mechanism induced by P. multocida strains of serotype A and D. [ABSTRACT FROM AUTHOR]

Abstract Background Prostate cancer is one of the most common and socially significant cancers among men. The aim of our study was to reveal changes in miRNA expression profiles associated with lymphatic dissemination in prostate cancer and to identify the most prominent miRNAs as potential prognostic markers for future studies. Methods High-throughput miRNA sequencing was performed for 44 prostate cancer specimens taken from Russian patients, with and without lymphatic dissemination (N1 – 20 samples; N0 – 24 samples). Results We found at least 18 microRNAs with differential expression between N0 and N1 sample groups: miR-182-5p, miR-183-5p, miR-96-5p, miR-25-3p, miR-93-5p, miR-7-5p, miR-615-3p, miR-10b, miR-1248 (N1-miRs; elevated expression in N1 cohort; p

Abstract Background MicroRNAs (miRNAs) regulate adipose tissue development, which are closely related to subcutaneous and intramuscular fat deposition and adipocyte differentiation. As an important economic and agricultural animal, rabbits have low adipose tissue deposition and are an ideal model to study adipose regulation. However, the miRNAs related to fat deposition during the growth and development of rabbits are poorly defined. Methods In this study, miRNA-sequencing and bioinformatics analyses were used to profile the miRNAs in rabbit perirenal adipose tissue at 35, 85 and 120 days post-birth. Differentially expressed (DE) miRNAs between different stages were identified by DEseq in R. Target genes of DE miRNAs were predicted by TargetScan and miRanda. To explore the functions of identified miRNAs, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Results Approximately 1.6 GB of data was obtained by miRNA-seq. A total of 987 miRNAs (780 known and 207 newly predicted) and 174 DE miRNAs were identified. The miRNAs ranged from 18 nt to 26 nt. GO enrichment and KEGG pathway analyses revealed that the target genes of the DE miRNAs were mainly involved in zinc ion binding, regulation of cell growth, MAPK signaling pathway, and other adipose hypertrophy-related pathways. Six DE miRNAs were randomly selected, and their expression profiles were validated by q-PCR. Conclusions This is the first report of the miRNA profiles of adipose tissue during different growth stages of rabbits. Our data provide a theoretical reference for subsequent studies on rabbit genetics, breeding and the regulatory mechanisms of adipose development.

1. Growth performance is significant for chickens. MicroRNAs (miRNAs) have been found to play important roles in the post-transcriptional regulation of skeletal muscle growth. However, the mechanism of miRNAs in this process has not been elucidated. 2. This study involved collecting leg muscle from slow- and fast-growing groups of Bian chicken at 16 weeks of age for high-throughput sequencing. A total of 42 differentially expressed miRNAs (DEMs) were identified. Among them, 22 DEMs were up-regulated and 20 DEMs were down-regulated. 3. Biological process terms, relating to growth, were found by GO enrichment for target genes of DEMs and KEGG pathway analysis of target genes. This revealed some significantly enriched pathways closely related to skeletal muscle development, such as the calcium signalling pathway, ECM-receptor interaction, lysine degradation, apoptosis and tight junctions. Network interaction analysis of DEMs and target genes showed that the top fifty hub genes were targeted by thirteen DEMs. 4. Four important miRNAs (novel_miR_158, novel_miR_144, novel_miR_291, and miR-205a) as well as some other valuable miRNAs, such as gga-miR-214 and gga-miR-3525 were identified. The qPCR results of five DEMs were highly consistent with that of sequencing between the two groups, which proved the reliability of miRNA-seq. 5. The study will help to improve the molecular mechanism of miRNAs in chickens and guide future experiments concerning miRNA function in chicken growth. [ABSTRACT FROM AUTHOR]

Overexpression of growth hormone (GH) in gh -transgenic zebrafish of a highly studied lineage F0104 has earlier been reported to cause increased muscle growth. In addition to this, GH affects a broad range of cellular processes in transgenic fish, such as morphology, physiology, and behavior. Reports show changes such as decreased sperm quality and reduced reproductive performance in transgenic males. It is hypothesized that microRNAs are directly involved in the regulation of fertility potential during spermatogenesis. The primary aim of our study was to verify whether gh overexpression disturbs the sperm miRNA profile and influences the sperm quality in transgenic zebrafish. We report a significant increase in body weight of gh -transgenic males along with associated reduced sperm motility and other kinetic parameters in comparison to the non-transgenic group. MicroRNA transcriptome sequencing of gh -transgenic zebrafish sperms revealed expressions of 186 miRNAs, among which six miRNA were up-regulated (miR-146b, miR-200a-5p, miR-146a, miR-726, miR-184, and miR-738) and sixteen were down-regulated (miR-19d-3p, miR-126a-5p, miR-126b-5p, miR-22a-5p, miR-16c-5p, miR-20a-5p, miR-126b-3p, miR-107a-3p, miR-93, miR-2189, miR-202–5p, miR-221–3p, miR-125a, miR-125b-5p, miR-126a-3p, and miR-30c-5p) in comparison to non-transgenic zebrafish. Some of the dysregulated miRNAs were previously reported to be related to abnormalities in sperm quality and reduced reproduction ability in other species. In this study, an average of 134 differentially expressed miRNAs-targeted genes were predicted using the in silico approach. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that the genes of affected pathways were primarily related to spermatogenesis, sperm motility, and cell apoptosis. Our results suggested that excess GH caused a detrimental effect on sperm microRNAome, consequently reducing the sperm quality and reproductive potential of zebrafish males. [ABSTRACT FROM AUTHOR]

17α-Methyltestosterone (MT) is a synthetic androgen. The objective of this study was to explore the effects of exogenous MT on the growth and gonadal development of female rare minnow Gobiocypris rarus. Female G. rarus groups were exposed to 25–100 ng/L of MT for 7 days. After exposure for 7 days, the total weight and body length were significantly decreased in the 50-ng/L MT groups. The major oocytes in the ovaries of the control group were vitellogenic oocytes (Voc) and cortical alveolus stage oocytes (Coc). In the MT exposure groups, some fish had mature ovaries with a relatively lower proportion of mature oocytes, and the diameter of the perinucleolar oocytes (Poc) was decreased compared with those of the control group. Ovarian VTG, FSH, LH, 11-KT, E2, and T were significantly increased after exposure to 50 ng/L of MT for 7 days. Unigenes (73,449), 24 known mature microRNAs (miRNAs), and 897 novel miRNAs in the gonads of G. rarus were found using high-throughput sequencing. Six mature miRNAs (miR-19, miR-183, miR-203, miR-204, miR-205, and miR-96) as well as six differentially expressed genes (fabp3 , mfap4 , abca1 , foxo3 , tgfb1 , and zfp36l1) that may be associated with ovarian development and innate immune response were assayed using qPCR. Furthermore, the miR-183 cluster and miR-203 were differentially expressed in MT-exposed ovaries of the different G. rarus groups. This study provides some information about the role of miRNA–mRNA pairs in the regulation of ovarian development and innate immune system, which will facilitate future studies of the miRNA–RNA-associated regulation of teleost reproduction. [ABSTRACT FROM AUTHOR]

As we have suggested, epigenetic factors, such as microRNAs (miRNAs), can interact with genetic programs to regulate B cell functions, thereby informing antibody and autoantibody responses. We have shown that histone deacetylase (HDAC) inhibitors (HDI) inhibit the differentiation events critical to the maturation of the antibody response: class-switch DNA recombination (CSR), somatic hypermutation (SHM), and plasma cell differentiation, by modulating intrinsic B cell mechanisms. HDI repress the expression of AID and Blimp-1, which are critical for CSR/SHM and plasma cell differentiation, respectively, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm1 mRNAs, as demonstrated by multiple qRT-PCRs (J Immunol 193:5933-5950, 2014). To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed genome-wide miRNA-Seq and mRNA-Seq analysis in B cells stimulated by LPS plus IL-4 and treated with HDI or nil. Consistent with what we have shown using qRT-PCR, these HDI-treated B cells displayed reduced expression of Aicda and Prdm1, and increased expression of miR-155, miR-181b, and miR-361, which target Aicda, and miR-23b, miR-30a, and miR-125b, which target Prdm1. In B cells induced to undergo CSR and plasma cell differentiation, about 23% of over 22,000 mRNAs analyzed were expressed at a significantly high copy number (more than 20 copies/cell). Only 18 (0.36%) of these highly expressed mRNAs, including Aicda, Prdm1, and Xbp1, were downregulated by HDI by 50% or more. Further, only 16 (0.30%) of the highly expressed mRNAs were upregulated (more than twofold) by HDI. The selectivity of HDI-mediated modulation of gene expression was emphasized by unchanged expression of the genes that are involved in regulation, targeting, or DNA repair processes of CSR, as well as unchanged expression of the genes encoding epigenetic regulators and factors that are important for cell signaling or apoptosis. Our findings indicate that, in B cells induced to undergo CSR and plasma cell differentiation, HDI modulate selected miRNAs and mRNAs, possibly as a result of HDACs existing in unique contexts of HDAC/cofactor complexes, as occurring in B lymphocytes, particularly when in an activated state.

Background: Improved prognostication of oropharyngeal squamous cell carcinoma (OPSCC) may facilitate individualized patient management. The goal of this study was to develop and validate a prognostic signature based on microRNA sequencing (miRNA-seq) analysis.Methods: We collected tumor specimens for miRNA-seq analysis from OPSCC patients treated at Washington University in St Louis (n = 324) and Vanderbilt University (n = 130). OPSCC patients (n = 79) from The Cancer Genome Atlas Program were also included for independent validation. Univariate and multivariable Cox regression analyses were performed to identify miRNAs associated with disease outcomes. All statistical tests were 2-sided.Results: By miRNA-seq profiling analysis, we identified a 26-miRNA signature. Based on computed risk scores of the signature, we classified the patients into low- and high-risk groups. In the training cohort, the high-risk group had much shorter overall survival compared with the low-risk group (hazard ratio [HR] = 3.80, 95% confidence interval [CI] = 2.37 to 6.10, P < .001). Subgroup analysis further revealed that the signature was prognostic for HPV-positive OPSCCs (HR = 3.07, 95% CI = 1.65 to 5.71, P < .001). Multivariable analysis indicated that the signature was independent of common clinicopathologic factors for OPSCCs. Importantly, the miRNA signature was a statistically significant predictor of overall survival in independent validation cohorts (The Cancer Genome Atlas Program cohort: HR = 6.05, 95% CI = 2.10 to 17.37, P < .001; Vanderbilt cohort: HR = 7.98, 95% CI = 3.99 to 15.97, P < .001; Vanderbilt HPV-positive cohort: HR = 8.71, 95% CI = 2.70 to 28.14, P < .001).Conclusions: The miRNA signature is a robust and independent prognostic tool for risk stratification of OPSCCs including HPV-positive OPSCCs. [ABSTRACT FROM AUTHOR]

Thermosensitive cytoplasmic male sterile (TCMS) lines play an important role in wheat breeding, heterosis utilization, and germplasm innovation. MicroRNAs (miRNAs) can regulate the expression level of target genes by inhibiting the translation of these genes. YS3038 is a wheat TCMS line. In this study, the fertility conversion mechanism of YS3038 was studied by examining the abortion characteristics of YS3038, the regulation pattern of miRNAs and the target genes of miRNAs in YS3038. MiRNA-seq was performed on three important stages of YS3038 under sterile and fertile conditions. Then, the clean reads were aligned with some databases to filter other ncRNAs and repeats. The known miRNAs and novel miRNAs were predicted by sequence comparison with known miRNAs from miRbase. Differential expression of miRNAs between different stages and between different fertile conditions was analyzed, and functional analysis of target genes with opposite expression patterns as those of the miRNAs was conducted. The Ubisch bodies and microspores of sterile anthers were covered with filamentous materials. The degradation of the tapetum cells, the chloroplast structure of endothecium cells, and the microspore structure were abnormal. Microspore development was hindered from the late uninucleate stage to the binucleate stage. Twenty, 52, and 68 differentially expressed miRNAs (DEmiRs) were identified at the early uninucleate, late uninucleate, and binucleate stages, respectively, and there were 0, 7, and 72 differentially expressed target genes (DETGs), respectively, at these three stages. At the binucleate stage, 29 DEmiRs had 41 target mRNAs in total, and the expression patterns of the 41 target mRNAs were opposite to those of the 29 miRNAs. Fifteen significantly enriched KEGG pathways were associated with the 41 target mRNAs. Leucine-rich repeat receptor-like kinases (LRR-RLKs) play important roles in plant developmental and physiological processes. Some studies have shown that the expression of LRR-RLKs is related to the differentiation of microsporocytes and tapetum cells and to male sterility. An LRR-RLK (TaeRPK) gene was silenced by the barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) method, and the seed setting rates of the TaeRPK -silenced plants (3.51%) were significantly lower than those of the negative control plants (88.78%) (P < 0.01). Thus, the TaeRPK gene is likely to be involved in the fertility conversion of YS3038. • The abnormal structure of Ubisch body, tapetum, and chloroplast caused the sterility of YS3038. • The microspore development was hindered from the late uninucleate stage to the binucleate stage. • The 41 target mRNAs with the opposite pattern of miRNAs were highly correlated with fertility conversion. • The TaeRPK gene was involved in the fertility conversion. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD), the leading form of dementia, is associated with abnormal tau and β-amyloid accumulation in the brain. We conducted a miRNA-seq study to identify miRNAs associated with AD in the post-mortem brain from the inferior frontal gyrus (IFG, n = 69) and superior temporal gyrus (STG, n = 81). Four and 64 miRNAs were differentially expressed (adjusted p -value < 0.05) in AD compared to cognitively normal controls in the IFG and STG, respectively. We observed down-regulation of several miRNAs that have previously been implicated in AD, including hsa-miR-212-5p and hsa-miR-132-5p, in AD samples across both brain regions, and up-regulation of hsa-miR-146a-5p, hsa-miR-501-3p, hsa-miR-34a-5p, and hsa-miR-454-3p in the STG. The differentially expressed miRNAs were previously implicated in the formation of amyloid-β plaques, the dysregulation of tau, and inflammation. We have also observed differential expressions for dozens of other miRNAs in the STG, including hsa-miR-4446-3p, that have not been described previously. Putative targets of these miRNAs (adjusted p -value < 0.1) were found to be involved in Wnt signaling pathway, MAPK family signaling cascades, sphingosine 1-phosphate (S1P) pathway, adaptive immune system, innate immune system, and neurogenesis. Our results support the finding of dysregulated miRNAs previously implicated in AD and propose additional miRNAs that appear to be dysregulated in AD for experimental follow-up. [ABSTRACT FROM AUTHOR]

While several microRNAs (miRNAs) that regulate the endotheliogenesis and further promote angiogenesis have been identified in various cancers, the identification of miRNAs that can drive the differentiation of adipose derived stromal/stem cells (ASCs) into the endothelial lineage has been largely unexplored. In this study, CD34+ ASCs sorted using magnetic bead separation were induced to differentiate along the endothelial pathway. miRNA sequencing of ASCs at day 3, 9, and 14 of endothelial differentiation was performed on Ion Proton sequencing system. The data obtained by this high-throughput method were aligned to the human genome HG38, and the differentially expressed miRNAs during endothelial differentiation at various time points (day 3, 9, and 14) were identified. The gene targets of the identified miRNAs were obtained through miRWalk database. The network-pathway analysis of miRNAs and their targets was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools to determine the potential candidate miRNAs that promote endothelial differentiation. Based on these analyses, six upregulated miRNAs (miR-181a-5p, miR-330-5p, miR-335-3p, miR-15b-5p, miR-99a-5p, and miR-199a-5p) and six downregulated miRNAs (miR-145-5p, miR-155-5p, miR-193a-3p, miR-125a-5p, miR-221-5p, and miR-222-3p) were chosen for further studies. In vitro evaluation of these miRNAs to induce endothelial differentiation when transfected into CD34+ sorted ASCs was studied using Von Willebrand Factor (VWF) staining and quantitative real time–polymerase chain reaction (qRT-PCR). Our results suggest that miRNAs: 335-5p, 330-5p, 181a-5p and anti-miRNAs: 125a-5p, 145-5p can likely induce endothelial differentiation in CD34+ sorted ASCs. Further studies are clearly required to elucidate the specific mechanisms on how miRNAs or anti-miRNAs identified through bioinformatics approach can induce the endotheliogenesis in ASCs. [ABSTRACT FROM AUTHOR]

Background: The domestication of tree shrews represents an important advance in the development of standardized laboratory animals. Little is known regarding the miRNA changes that accompany the transformation of wild tree shrews into domestic tree shrews. Results: By performing miRNA-seq analysis on wild and domestic tree shrews, we identified 2410 miRNAs and 30 differentially expressed miRNAs in the hippocampus during tree shrew domestication. A KEGG analysis of the differentially expressed genes showed that the differentially expressed miRNAs were associated with ECM-receptor interaction, the phosphatidylinositol signaling system, protein digestion and absorption, inositol phosphate metabolism, lysine degradation, fatty acid degradation and focal adhesion. Most of these pathways could be classified under environmental information processing, organismal systems and metabolism. The miRNAs exclusively expressed in wild and tame tree shrews GO enriched in terms of divergent functions. The miRNA-mRNA networks suggested that novel-m1388-5p and novel-m0746-5p might play regulatory roles in domestication of tree shrews. Real-time RT-PCR analysis was employed to verify the presence of these miRNAs. Conclusion: We identified a number of candidate miRNA-regulated domestication genes that may represent targets for selection during the domestication of tree shrews. [ABSTRACT FROM AUTHOR]

• Expression levels of miRNAs were determined in anterior cingulate cortex of MDD subjects by miRNA-seq. • A significant number of miRNAs were differentially regulated in MDD subjects. • Predicted target-based gene ontology suggested that altered miRNAs may be related to abnormalities in neuronal functions, atrophy and volume loss in ACC of MDD subjects. MicroRNAs (miRNAs), a family of non-coding RNAs, have recently gained a considerable attention in neuropsychiatric disorders. Being a pleiotropic modulator of target gene(s), miRNA has been recognized as central to downstream gene regulatory networks. In the recent past, reports have suggested their role in changing the epigenetic landscape in brain of subjects with major depressive disorder (MDD). Anterior cingulate cortex (ACC) is a brain area implicated in several complex cognitive functions, such as impulse control, emotion, and decision-making and is associated with psychopathology associated with mood regulation. In this study, we examined whether MDD is associated with altered miRNA transcriptome in ACC and whether altered miRNA landscape is associated with modifications in specific gene network(s) at the functional level. Using next generation sequencing (NGS), it was observed that 117 miRNAs (4.61%) were significantly upregulated and 54 (2.13%) were downregulated in MDD subjects (n = 22) compared with non-psychiatric controls (n = 25). Using 24 most significantly upregulated miRNAs in the MDD group, we determined functional enrichment of target genes and found them to be associated with long-term potentiation, neurotrophin signaling, and axon guidance. Intra- and inter-cluster similarities of enriched terms based on overrepresented gene list showed neurobiological functions associated with neuronal growth and survival. Web centric parameters and ontology enrichment functions identified two major domains related to phosphatidyl signaling, GTPase signaling, neuronal migration, and neurotrophin signaling. Our findings of altered miRNA landscape along with a shift in targetome relate to previously reported morphometric changes and neuronal atrophy in ACC of MDD subjects. [ABSTRACT FROM AUTHOR]

Abstract Background Despite diverging levels of amyloid-β (Aβ) and TAU pathology, different mouse models, as well as sporadic AD patients show predictable patterns of episodic memory loss. MicroRNA (miRNA) deregulation is well established in AD brain but it is unclear whether Aβ or TAU pathology drives those alterations and whether miRNA changes contribute to cognitive decline. Methods miRNAseq was performed on cognitively intact (4 months) and impaired (10 months) male APPtg (APPswe/PS1L166P) and TAUtg (THY-Tau22) mice and their wild-type littermates (APPwt and TAUwt). We analyzed the hippocampi of 12 mice per experimental group (n = 96 in total), and employed a 2-way linear model to extract differentially expressed miRNAs. Results were confirmed by qPCR in a separate cohort of 4 M and 10 M APPtg and APPwt mice (n = 7–9 per group) and in human sporadic AD and non-demented control brain. Fluorescent in situ hybridization identified their cellular expression. Functional annotation of predicted targets was performed using GO enrichment. Behavior of wild-type mice was assessed after intracerebroventricular infusion of miRNA mimics. Results Six miRNAs (miR-10a-5p, miR-142a-5p, miR-146a-5p, miR-155-5p, miR-211-5p, miR-455-5p) are commonly upregulated between APPtg and TAUtg mice, and four of these (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) are altered in AD patients. All 6 miRNAs are strongly enriched in neurons. Upregulating these miRNAs in wild-type mice is however not causing AD-related cognitive disturbances. Conclusion Diverging AD-related neuropathologies induce common disturbances in the expression of neuronal miRNAs. 4 of these miRNAs are also upregulated in AD patients. Therefore these 4 miRNAs (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) appear part of a core pathological process in AD patients and APPtg and TAUtg mice. They are however not causing cognitive disturbances in wild-type mice. As some of these miRNA target AD relevant proteins, they may be, in contrast, part of a protective response in AD.

Background: MicroRNAs (miRNAs) regulate adipose tissue development, which are closely related to subcutaneous and intramuscular fat deposition and adipocyte differentiation. As an important economic and agricultural animal, rabbits have low adipose tissue deposition and are an ideal model to study adipose regulation. However, the miRNAs related to fat deposition during the growth and development of rabbits are poorly defined. Methods: In this study, miRNA-sequencing and bioinformatics analyses were used to profile the miRNAs in rabbit perirenal adipose tissue at 35, 85 and 120 days post-birth. Differentially expressed (DE) miRNAs between different stages were identified by DEseq in R. Target genes of DE miRNAs were predicted by TargetScan and miRanda. To explore the functions of identified miRNAs, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Results: Approximately 1.6 GB of data was obtained by miRNA-seq. A total of 987 miRNAs (780 known and 207 newly predicted) and 174 DE miRNAs were identified. The miRNAs ranged from 18 nt to 26 nt. GO enrichment and KEGG pathway analyses revealed that the target genes of the DE miRNAs were mainly involved in zinc ion binding, regulation of cell growth, MAPK signaling pathway, and other adipose hypertrophy-related pathways. Six DE miRNAs were randomly selected, and their expression profiles were validated by q-PCR. Conclusions: This is the first report of the miRNA profiles of adipose tissue during different growth stages of rabbits. Our data provide a theoretical reference for subsequent studies on rabbit genetics, breeding and the regulatory mechanisms of adipose development. [ABSTRACT FROM AUTHOR]

Red skin color in Plectropomus leopardus is important to its ornamental and economic value. However, the color of P. leopardus can change during the rearing process, darkening and turning black due to the influence of environmental background color. The underlying molecular mechanisms that regulate this phenomenon remain unclear. MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that play important roles in numerous biological processes, such as skin differentiation and color formation in many animals. Therefore, we performed miRNA sequencing of P. leopardus skin before (initial) and after rearing with three different background colors (white, black, and blue) using Illumina sequencing to identify candidate miRNAs that may contribute to skin color differentiation. In total, 154,271,376 clean reads were obtained, with over 92 % of them successfully mapped to the P. leopardus reference genome. The miRNA length distributions of all samples displayed peaks around a typical length of 22 nt. Within these sequences, 243 known and 287 novel miRNAs were identified. A total of 65 significantly differentially expressed miRNAs (DEMs) were identified (P < 0.05), including 40 known DEMs and 25 novel DEMs. These DEMs included novel_561, miR-141-3p, and miR-129-5p, whose target genes were primarily associated with pigmentation related processes, including tyrosine metabolism, melanogenesis, and the Wnt signaling pathway. These findings shed light on the potential roles of miRNAs in the darkening of skin color in P. leopardus , thus enhancing our understanding of the molecular mechanisms involved in skin pigmentation differentiation in this species. [Display omitted] • A total of 65 significantly DEMs were identified (P < 0.05) in different groups. • Novel_561, miR-141-3p, and miR-129-5p were involved in pigmentation. • These findings shed light on the potential roles of miRNAs in the darkening of skin color in P. leopardus. [ABSTRACT FROM AUTHOR]

Temperature is a critical factor that regulates the reproduction processes in teleost. However, the gonadal response mechanism to cold stress in fish remains largely unknown. In the present study, female zebrafish were exposed to different extents of low temperatures at 18 °C and 10 °C for 48 h. The ovarian histology was remarkably damaged after cold stress exposure. Integrated analysis of miRNA-mRNA was used to investigate the ovarian response to acute cold stress. A large number of mRNAs and miRNAs were altered by cold stress, which are involved in extensive biological processes. It is indicated that the signal transduction of MAPK and Calcium signaling pathway is highly engaged in zebrafish ovary to adapt to cold stress. The immune system was dysregulated by cold stress while the ovarian autophagy was activated. Remarkably increased gene number related to reproductive functions was identified in the cold stress at 10 °C compared to the control. The cold stress-induced dysregulated reproductive genes include star , hsd3b1 , hsd17b1 , inha , insl3 , amh , nanos1 and foxl2. Combined with the dysregulated insulin, IGF and progesterone signaling, it is suggested that cold stress affects ovarian function in multiple aspects, including oocyte meiosis, folliculogenesis, final maturation and ovarian maintenance. On the other hand, the ovarian miRNA-mRNA regulatory network response to cold stress was also constructed. Overall, our result revealed the ovarian response to cold stress in zebrafish and provided insight into the fish adaptation mechanism to acute temperature change. [Display omitted] • Ovarian response to cold stress was investigated by miRNA-mRNA analysis. • Cold stress altered ovarian signal transduction, immune system, and autophagy. • Oocyte maturation was inhibited by defects on insulin and progesterone signaling. • Cold stress dysregulate ovarian function from multiple aspects. [ABSTRACT FROM AUTHOR]

Disc degeneration is a common clinical condition in which damaged discs cause chronic pain; however, a laboratory diagnosis method for its detection is not available. As circulating miRNAs have potential as biomarkers, their application in disc degeneration has not been explored. Here, we prepared serum miRNAs from a mouse disc degeneration model and performed miRNA‐Seq and quantitative PCR to characterize disc degeneration–associated miRNAs. We identified three miRNAs, including miR‐26a‐5p, miR‐122‐5p and miR‐215‐5p, undergoing perturbation during the pathogenesis of disc degeneration. Specifically, the levels of miR‐26a‐5p in the serum demonstrated steady increases in the model of disc degeneration, compared with those in the pre‐injury samples of younger age or compared with normal controls of the same age but without disc degeneration, whereas the miRNAs miR‐122‐5p and miR‐215‐5p exhibited lower expression in post‐injury samples than in their counterparts without the surgery. Moreover, we found that miR‐26a‐5p targets Smad1 expression, and Smad1 negatively regulates Vegfa expression in disc cells, and thus, miR‐26a‐5p promotes disc degeneration. In summary, we established a method that consistently profiles circulating miRNAs and identified multiple miRNAs as promising biomarkers for disc degeneration, among which miR‐26a‐5p enhances VEGF expression during disc degeneration through targeting Smad1 signalling. [ABSTRACT FROM AUTHOR]

Host genetic factors play an important role in diverse host outcomes after influenza A (H7N9) infection. Studying differential responses of inbred mouse lines with distinct genetic backgrounds to influenza virus infection could substantially increase our understanding of the contributory roles of host genetic factors to disease severity. Here, we utilized an integrated approach of mRNA-seq and miRNA-seq to investigate the transcriptome expression and regulation of host genes in C57BL/6J and DBA/2J mouse strains during influenza virus infection. The differential pathogenicity of influenza virus in C57BL/6J and DBA/2J has been fully demonstrated through immunohistochemical staining, histopathological analyses, and viral replication assessment. A transcriptional molecular signature correlates to differential host response to infection has been uncovered. With the introduction of temporal expression pattern analysis, we demonstrated that host factors responsible for influenza virus replication and host-virus interaction were significantly enriched in genes exhibiting distinct temporal dynamics between different inbred mouse lines. A combination of time-series expression analysis and temporal expression pattern analysis has provided a list of promising candidate genes for future studies. An integrated miRNA regulatory network from both mRNA-seq and miRNA-seq revealed several regulatory modules responsible for regulating host susceptibilities and disease severity. Overall, a comprehensive framework for analyzing host susceptibilities to influenza infection was established by integrating mRNA-seq and miRNA-seq data of inbred mouse lines. This work suggests novel putative molecular targets for therapeutic interventions in seasonal and pandemic influenza. [ABSTRACT FROM AUTHOR]

Next-generation sequencing was used to analyze the effects of toxic microcystin-LR (MC-LR) on silver carp (Hypophthalmichthys molitrix). Silver carps were intraperitoneally injected with MC-LR, and RNA-seq and miRNA-seq in the liver were analyzed at 0.25, 0.5, and 1 h. The expression of glutathione S-transferase (GST), which acts as a marker gene for MC-LR, was tested to determine the earliest time point at which GST transcription was initiated in the liver tissues of the MC-LR-treated silver carps. Hepatic RNA-seq/miRNA-seq analysis and data integration analysis were conducted with reference to the identified time point. Quantitative PCR (qPCR) was performed to detect the expression of the following genes at the three time points: heme oxygenase 1 (HO-1), interleukin-10 receptor 1 (IL-10R1), apolipoprotein A-I (apoA-I), and heme binding protein 2 (HBP2). Results showed that the liver GST expression was remarkably decreased at 0.25 h (P < 0.05). RNA-seq at this time point revealed that the liver tissue contained 97,505 unigenes, including 184 significantly different unigenes and 75 unknown genes. Gene Ontology (GO) term enrichment analysis suggested that 35 of the 145 enriched GO terms were significantly enriched and mainly related to the immune system regulation network. KEGG pathway enrichment analysis showed that 18 of the 189 pathways were significantly enriched, and the most significant was a ribosome pathway containing 77 differentially expressed genes. miRNA-seq analysis indicated that the longest miRNA had 22 nucleotides (nt), followed by 21 and 23 nt. A total of 286 known miRNAs, 332 known miRNA precursor sequences, and 438 new miRNAs were predicted. A total of 1,048,575 mRNA-miRNA interaction sites were obtained, and 21,252 and 21,241 target genes were respectively predicted in known and new miRNAs. qPCR revealed that HO-1, IL-10R1, apoA-I, and HBP2 were significantly differentially expressed and might play important roles in the toxicity and liver detoxification of MC-LR in fish. These results were consistent with those of high-throughput sequencing, thereby verifying the accuracy of our sequencing data. RNA-seq and miRNA-seq analyses of silver carp liver injected with MC-LR provided valuable and new insights into the toxic effects of MC-LR and the antitoxic mechanisms of MC-LR in fish. [ABSTRACT FROM AUTHOR]

Notch signaling as a conserved cell fate regulator is involved in the regulation of cell quiescence, proliferation, differentiation and postnatal tissue regeneration. However, how Notch signaling regulates porcine satellite cells (PSCs) has not been elucidated. We stably transfected Notch1 intracellular domain (N1ICD) into PSCs to analyze the gene expression profile and miRNA-seq. The analysis of the gene expression profile identified 295 differentially-expressed genes (DEGs) in proliferating-N1ICD PSCs (P-N1ICD) and nine DEGs on differentiating-N1ICD PSCs (D-N1ICD), compared with that in control groups (P-Control and D-Control, respectively). Analyzing the underlying function of DEGs showed that most of the upregulated DEGs enriched in P-N1ICD PSCs are related to the cell cycle. Forty-four and 12 known differentially-expressed miRNAs (DEMs) were identified in the P-N1ICD PSCs and D-N1ICD PSCs group, respectively. Furthermore, we constructed the gene-miRNA network of the DEGs and DEMs. In P-N1ICD PSCs, miR-125a, miR-125b, miR-10a-5p, ssc-miR-214, miR-423 and miR-149 are downregulated hub miRNAs, whose corresponding hub genes are marker of proliferation Ki-67 (MKI67) and nuclear receptor binding SET domain protein 2 (WHSC1). By contrast, miR-27a, miR-146a-5p and miR-221-3p are upregulated hub miRNAs, whose hub genes are RUNX1 translocation partner 1 (RUNX1T1) and fibroblast growth factor 2 (FGF2). All the hub miRNAs and genes are associated with cell proliferation. Quantitative RT-PCR results are consistent with the gene expression profile and miRNA-seq results. The results of our study provide valuable information for understanding the molecular mechanisms underlying Notch signaling in PSCs and skeletal muscle development. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) patients who do not achieve landmark responses following treatment with imatinib mesylate (IM) are considered IM-resistant. Although IM-resistance can be due to BCR-ABL kinase domain (KD) mutations, many IM-resistant patients do not have detectable BCR-ABL KD mutations. MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression. To investigate the role of miRNAs in IM-resistance, we recruited 8 chronic phase CML patients with IM-resistance who tested negative for BCR-ABL KD mutations and 2 healthy normal controls. Using miRNA sequencing, we identified 54 differentially expressed miRNAs; 43 of them downregulated. The 3 most differentially downregulated miRNAs were miR-146a-5p, miR-99b-5p and miR-151a-5p. Using real-time quantitative reverse transcriptase-polymerase chain reaction, the expression patterns of the 3 miRNAs were validated on the same cohort of 8 patients in addition to 3 other IM-resistant CML patients. In-silico analysis showed that the predicted gene targets are ATRIP, ATR, WDR48, RAD51C and FANCA genes which are involved in the Fanconi Anemia/BRCA pathway. This pathway regulates DNA damage response (DDR) and influences disease response to chemotherapy. Thus it is conceivable that DDR constitutes a key component in IM-resistance. Further research is needed to elucidate miRNA modulation of the predicted gene targets. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are one family of small noncoding RNAs that function to modulate the activity of specific mRNA targets in animals. To understand the role of miRNAs in regulating genes involved in the host immune response to RNA viruses, we profiled and characterized the miRNAs of swine peripheral blood mononuclear cells (PBMC) stimulated with poly I:C, a synthetic dsRNA analog, by miRNA-sequencing (miRNA-seq). We identified a total of 905 miRNAs, of which 503 miRNAs were firstly exploited herein with no annotation in the latest miRBase 21.0. Expression analysis demonstrated that poly I:C stimulation can elicit significantly differentially expressed (DE) miRNAs in Dapulian (n = 20), one Chinese indigenous breed, as well as Landrace (n = 23). By integrating the mRNA expression profiles of the same sample with miRNA profiles, we carried out function analyses of the target genes of these DE miRNAs, with the results indicating that target genes were most enriched in some immune-related pathways and gene ontology (GO) terms, suggesting that DE miRNAs play an important role in the regulation of host to poly I:C stimulation. Furthermore, we also detected 43 and 61 significantly DE miRNAs between the two breeds in the control sample groups and poly I:C stimulation groups, respectively, which may be involved in regulation of the different characteristics of the two breeds. This study describes for the first time the PBMC miRNA transcriptomic response to poly I:C stimulation in pigs, which not only contributes to a broad view of the pig miRNAome but improves our understanding of miRNA function in regulating host immune response to RNA viruses. [ABSTRACT FROM AUTHOR]

At the core of the changes characteristic of alcoholism are alterations in gene expression in the brain of the addicted individual. These changes are believed to underlie some of the neuroadaptations that promote compulsive drinking. Unfortunately, the mechanisms by which alcohol consumption produces changes in gene expression remain poorly understood. MicroRNAs (miRNAs) have emerged as important regulators of gene expression because they can coordinately modulate the translation efficiency of large sets of specific mRNAs. Here, we investigate the early miRNA responses elicited by an acute sedating dose of alcohol in theDrosophilamodel organism. In our analysis, we combine the power of next-generation sequencing withDrosophilagenetics to identify alcohol-sensitive miRNAs and to functionally test them for a role in modulating alcohol sensitivity. We identified 14 knownDrosophilamiRNAs, and 13 putative novel miRNAs that respond to an acute sedative exposure to alcohol. Using the GeneSwitch Gal4/UAS system, a subset of these ethanol-responsive miRNAs was functionally tested to determine their individual contribution in modulating ethanol sensitivity. We identified two microRNAs that when overexpressed significantly increased ethanol sensitivity: miR-6 and miR-310. MicroRNA target prediction analysis revealed that the different alcohol-responsive miRNAs target-overlapping sets of mRNAs. Alcoholism is the product of accumulated cellular changes produced by chronic ethanol consumption. Although all of the changes described herein are extremely rapid responses evoked by a single ethanol exposure, understanding the gene expression changes that occur in the first few minutes after ethanol exposure will help us to categorize ethanol responses into those that are near instantaneous and those that are emergent responses produced only by repeated ethanol exposure. [ABSTRACT FROM AUTHOR]

High-throughput sequencing of miRNAs has revealed the diversity and variability of mature and functional short noncoding RNAs, including their genomic origins, biogenesis pathways, sequence variability, and newly identified products such as miRNA-offset RNAs (moRs). Here we review known cases of alternative mature miRNA-like RNA fragments and propose a revised definition of miRNAs to encompass this diversity. We then review nomenclature guidelines for miRNAs and propose to extend nomenclature conventions to align with those for protein-coding genes established by international consortia. Finally, we suggest a system to encompass the full complexity of sequence variations (i.e., isomiRs) in the analysis of small RNA sequencing experiments. [ABSTRACT FROM AUTHOR]

Although the accuracy of detecting the expression of miRNAs by quantitative real-time polymerase chain reaction (qRT-PCR) is highly dependent on reliable reference miRNAs, many commonly used reference miRNAs are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. To solve this problem, we analyzed the global expression profiles of thousands of samples in 14 types of common human tumors released by The Cancer Genome Atlas (TCGA), and identified the most stably and highly expressed miRNAs as candidate reference miRNAs in each type of tumor. We found that miR-361-5p and let-7i-5p were the most recommended candidate reference miRNAs in nine and eight types of tumors, respectively, followed by let-7a-5p, mir-28-5p and miR-99b-5p. Our results are of important value to those researchers focused on miRNA; however, these candidate reference miRNAs still need to be validated prior to their use in qRT-PCR studies. [ABSTRACT FROM AUTHOR]

Background: We carried out a genome-wide study, using microRNA sequencing (miRNA-seq), aimed at identifying miRNAs in primary sensory neurons that are associated with neuropathic pain. Such scans usually yield long lists of transcripts regulated by nerve injury, but not necessarily related to pain. To overcome this we tried a novel search strategy: identification of transcripts regulated differentially by nerve injury in rat lines very similar except for a contrasting pain phenotype. Dorsal root ganglia (DRGs) L4 and 5 in the two lines were excised 3 days after spinal nerve ligation surgery (SNL) and small RNAs were extracted and sequenced. Results: We identified 284 mature miRNA species expressed in rat DRGs, including several not previously reported, and 3340 unique small RNA sequences. Baseline expression of miRNA was nearly identical in the two rat lines, consistent with their shared genetic background. In both lines many miRNAs were nominally up- or down-regulated following SNL, but the change was similar across lines. Only 3 miRNAs that were expressed abundantly (rno-miR-30d-5p, rno-miR-125b-5p) or at moderate levels (rno-miR-379-5p) were differentially regulated. This makes them prime candidates as novel PNS determinants of neuropathic pain. The first two are known miRNA regulators of the expression of Tnf, Bdnf and Stat3, gene products intimately associated with neuropathic pain phenotype. A few non-miRNA, small noncoding RNAs (sncRNAs) were also differentially regulated. Conclusions: Despite its genome-wide coverage, our search strategy yielded a remarkably short list of neuropathic pain-related miRNAs. As 2 of the 3 are validated regulators of important pro-nociceptive compounds, it is likely that they contribute to the orchestration of gene expression changes that determine individual variability in pain phenotype. Further research is required to determine whether some of the other known or predicted gene targets of these miRNAs, or of the differentially regulated non-miRNA sncRNAs, also contribute. [ABSTRACT FROM AUTHOR]