ExosomePurity: tumour purity deconvolution in serum exosomes based on miRNA signatures.

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).

A heavy-tailed model for analyzing miRNA-seq raw read counts.

This article addresses the limitations of existing statistical models in analyzing and interpreting highly skewed miRNA-seq raw read count data that can range from zero to millions. A heavy-tailed model using discrete stable distributions is proposed as a novel approach to better capture the heterogeneity and extreme values commonly observed in miRNA-seq data. Additionally, the parameters of the discrete stable distribution are proposed as an alternative target for differential expression analysis. An R package for computing and estimating the discrete stable distribution is provided. The proposed model is applied to miRNA-seq raw counts from the Norwegian Women and Cancer Study (NOWAC) and the Cancer Genome Atlas (TCGA) databases. The goodness-of-fit is compared with the popular Poisson and negative binomial distributions, and the discrete stable distributions are found to give a better fit for both datasets. In conclusion, the use of discrete stable distributions is shown to potentially lead to more accurate modeling of the underlying biological processes.

Breed-Related Differential microRNA Expression and Analysis of Colostrum and Mature Milk Exosomes in Bamei and Landrace Pigs.

Breast milk, an indispensable source of immunological and nutrient components, is essential for the growth and development of newborn mammals. MicroRNAs (miRNAs) are present in various tissues and body fluids and are selectively packaged inside exosomes, a type of membrane vesicle. Milk exosomes have potential regulatory effects on the growth, development, and immunity of newborn piglets. To explore the differences in milk exosomes related to the breed and milk type, we isolated exosomes from colostrum and mature milk from domestic Bamei pigs and foreign Landrace pigs by using density gradient centrifugation and then characterized them by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Furthermore, the profiles and functions of miRNAs in the two types of pig milk exosomes were investigated using miRNA-seq and bioinformatics analysis. We identified a total of 1081 known and 2311 novel miRNAs in pig milk exosomes from Bamei and Landrace pigs. These differentially expressed miRNAs (DE-miRNAs) are closely associated with processes such as cell signaling, cell physiology, and immune system development. Functional enrichment analysis showed that DE-miRNA target genes were significantly enriched in endocytosis, the T cell receptor signaling pathway, and the Th17 cell differentiation signaling pathway. The exosomal miRNAs in both the colostrum and mature milk of the two pig species showed significant differences. Based on related signaling pathways, we found that the colostrum of local pig breeds contained more immune-system-development-related miRNAs. This study provides new insights into the possible function of milk exosomal miRNAs in the development of the piglet immune system.

Imprinted Dlk1-Gtl2 cluster miRNAs are potential epigenetic regulators of lamb fur quality.

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.

miRNA-seq identification and clinical validation of CD138+ and circulating miR-25 in treatment response of multiple myeloma.

BACKGROUND: Despite significant advancements in multiple myeloma (MM) therapy, the highly heterogenous treatment response hinders reliable prognosis and tailored therapeutics. Herein, we have studied the clinical utility of miRNAs in ameliorating patients' management. METHODS: miRNA-seq was performed in bone marrow CD138+ plasma cells (PCs) of 24 MM and smoldering MM (sMM) patients to analyze miRNAs profile. CD138+ and circulating miR-25 levels were quantified using in house RT-qPCR assays in our screening MM/sMM cohort (CD138+ plasma cells n = 167; subcohort of MM peripheral plasma samples n = 69). Two external datasets (Kryukov et al. cohort n = 149; MMRF CoMMpass study n = 760) served as institutional-independent validation cohorts. Patients' mortality and disease progression were assessed as clinical endpoints. Internal validation was performed by bootstrap analysis. Clinical benefit was estimated by decision curve analysis. RESULTS: miRNA-seq highlighted miR-25 of CD138+ plasma cells to be upregulated in MM vs. sMM, R-ISS II/III vs. R-ISS I, and in progressed compared to progression-free patients. The analysis of our screening cohort highlighted that CD138+ miR-25 levels were correlated with short-term progression (HR = 2.729; p = 0.009) and poor survival (HR = 4.581; p = 0.004) of the patients; which was confirmed by Kryukov et al. cohort (HR = 1.878; p = 0.005) and MMRF CoMMpass study (HR = 1.414; p = 0.039) validation cohorts. Moreover, multivariate miR-25-fitted models contributed to superior risk-stratification and clinical benefit in MM prognostication. Finally, elevated miR-25 circulating levels were correlated with poor survival of MM patients (HR = 5.435; p = 0.021), serving as a potent non-invasive molecular prognostic tool. CONCLUSIONS: Our study identified miR-25 overexpression as a powerful independent predictor of poor treatment outcome and post-treatment progression, aiding towards modern non-invasive disease prognosis and personalized treatment decisions.

Integrative mRNA-miRNA interaction analysis associated with the immune response in the head kidney of rainbow trout (Oncorhynchus mykiss) after infectious hematopoietic necrosis virus infection.

Rainbow trout (Oncorhynchus mykiss) is an important cold-water fish widely cultivated in China. The frequent occurrence of viral diseases caused by infectious hematopoietic necrosis virus (IHNV) seriously restricted the healthy development of the rainbow trout farming industry. However, the immune defense mechanism induced by IHNV in rainbow trout has not been fully elucidated. In the present study, we detected mRNA and miRNA expression profiles in rainbow trout head kidney after IHNV infection using RNA-seq and identified key immune-related genes and miRNAs. The results showed that a total of 7486 genes and 277 miRNAs were differentially expressed, and numerous differentially expressed genes (DEGs) enriched in the immune-related pathways such as Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathway were significantly up-regulated, including LGP2, MDA5, TRIM25, IRF3, IRF7, TLR3, TLR7, TLR8, MYD88, and IFN1. Integration analysis identified six miRNAs (miR-141-y, miR-200-y, miR-144-y, miR-2188-y, miR-725-y, and miR-203-y) that target at least six key immune-related genes (TRIM25, LGP2, TLR3, TLR7, IRF3, and IRF7). Further, we verified selected immune-related mRNAs and miRNAs through qRT-PCR and confirmed the reliability of the RNA-seq results. These findings improve our understanding of the immune mechanism of rainbow trout infected with IHNV and provide basic data for future breeding for disease resistance in rainbow trout.

Differentiation of Atrial Fibrillation and Atrial Fibrillation-Associated Ischemic Stroke Based on Serum Exosome miRNA-Seq.

INTRODUCTION: Atrial fibrillation (AF) is the most common cardiac arrhythmia in the general population, and stroke is the most severe complication of AF. Exosomal miRNAs have been reported to be candidates as biomarkers for cardiovascular diseases, including AF and stroke. This study aimed to identify differentially expressed miRNAs (DEMs) in serum exosomes of AF and AF-associated ischemic stroke (AF-IS) patients and evaluate their potential in distinguishing AF and AF-IS patients. METHODS: Serum exosomes were isolated from 8 healthy individuals with sinus rhythm (SR controls), 8 AF patients, and 8 AF-IS patients. miRNA-seq was performed to identify DEMs, and qRT-PCR analysis was performed to confirm the sequencing results. A support vector machine (SVM) model was developed using Python to distinguish AF and AF-IS patients. RESULTS: 68 and 86 DEMs were identified in serum exosomes of AF patients compared to AF-IS patients and SR controls, respectively. Levels of miR-641 and miR-30e-5p were found significantly higher in AF-IS patients. The SVM model achieved an accuracy of 100%, with an area under curve of 1. CONCLUSIONS: The results indicated that miRNA expression profiles of serum exosomes in AF patients were distinct from those in AF-IS patients, and based on the distinction, AF and AF-IS patients can be distinguished.

Changes in miRNA expression in the lungs of pigs supplemented with different levels and forms of vitamin D.

BACKGROUND: Vitamin D is an immunomodulator, and its effects have been linked to many diseases, including the pathogenesis of cancer. However, the effect of vitamin D supplementation on the regulation of gene expression of the lungs is not fully understood. This study aims to determine the effect of the increased dose of cholecalciferol and a combination of cholecalciferol + calcidiol, as well as the replacement of cholecalciferol with calcidiol, on the miRNA profile of healthy swine lungs. METHODS AND RESULTS: The swine were long-term (88 days) supplemented with a standard dose (2000IU/kg) of cholecalciferol and calcidiol, the increased dose (3000 IU/kg) of cholecalciferol, and the cholecalciferol + calcidiol combination: grower: 3000 IU/Kg of vitamin D (67% of cholecalciferol and 33% of calcidiol), finisher 2500 IU/Kg of vitamin D (60% of cholecalciferol and 40% of calcidiol). Swine lung tissue was used for Next Generation Sequencing (NGS) of miRNA. Long-term supplementation with the cholecalciferol + calcidiol combination caused significant changes in the miRNA profile. They embraced altered levels of the expression of miR-150, miR-193, miR-145, miR-574, miR-340, miR-381, miR-148 and miR-96 (q-value < 0.05). In contrast, raising the dose of cholecalciferol only changed the expression of miR-215, and the total replacement of cholecalciferol with calcidiol did not significantly affect the miRNAome profile. CONCLUSIONS: The functional analysis of differentially expressed miRNAs suggests that the use of the increased dose of the cholecalciferol + calcidiol combination may affect tumorigenesis processes through, inter alia, modulation of gene regulation of the TGF- ß pathway and pathways related to metabolism and synthesis of glycan.

Comparative Analysis of miRNA-mRNA Regulation in the Testes of Gobiocypris rarus following 17α-Methyltestosterone Exposure.

17α-Methyltestosterone (17MT), a synthetic organic compound commonly found in sewage waters, can affect reproduction in aquatic animals, such as tilapia and yellow catfish. In the present study, male Gobiocypris rarus were exposed to 25, 50, and 100 ng/L of 17α-methyltestosterone (17MT) for 7 days. We first analyzed miRNA- and RNA-seq results to determine miRNA-target gene pairs and then developed miRNA-mRNA interactive networks after 17MT administration. Total weights, total lengths, and body lengths were not significantly different between the test groups and control groups. The paraffin slice method was applied to testes of G. rarus in the MT exposure and control groups. We found that there were more mature sperm (S) and fewer secondary spermatocytes (SSs) and spermatogonia (SGs) in the testes of control groups. As 17MT concentration increased, fewer and fewer mature sperm (S) were observed in the testes of male G. rarus. The results showed that FSH, 11-KT, and E2 were significantly higher in individuals exposed to 25 ng/L 17MT compared with the control groups. VTG, FSH, LH, 11-KT, and E2 were significantly lower in the 50 ng/L 17MT exposure groups compared to the control groups. VTG, FSH, LH, 11-KT, E2, and T were significantly lower in the groups exposed to 100 ng/L 17MT. High-throughput sequencing revealed 73,449 unigenes, 1205 known mature miRNAs, and 939 novel miRNAs in the gonads of G. rarus. With miRNA-seq, 49 (MT25-M vs. Con-M), 66 (MT50-M vs. Con-M), and 49 (MT100-M vs. Con-M) DEMs were identified in the treatment groups. Five mature miRNAs (miR-122-x, miR-574-x, miR-430-y, lin-4-x, and miR-7-y), as well as seven differentially expressed genes (soat2, inhbb, ihhb, gatm, faxdc2, ebp, and cyp1a1), which may be associated with testicular development, metabolism, apoptosis, and disease response, were assayed using qRT-PCR. Furthermore, miR-122-x (related to lipid metabolism), miR-430-y (embryonic development), lin-4-x (apoptosis), and miR-7-y (disease) were differentially expressed in the testes of 17MT-exposed G. rarus. This study highlights the role of miRNA-mRNA pairs in the regulation of testicular development and immune response to disease and will facilitate future studies on the miRNA-RNA-associated regulation of teleost reproduction.

Study of microRNA expression in Salmonella Typhimurium-infected porcine ileum reveals miR-194a-5p as an important regulator of the TLR4-mediated inflammatory response.

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.

Proteomic and miRNA Profiles of Exosomes Derived from Myometrial Tissue in Laboring Women.

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.

The Impact of the Anticoagulant Type in Blood Collection Tubes on Circulating Extracellular Plasma MicroRNA Profiles Revealed by Small RNA Sequencing.

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.

ALDH3A2, ODF2, QSOX2, and MicroRNA-503-5p Expression to Forecast Recurrence in TMPRSS2-ERG-Positive Prostate Cancer.

Following radical surgery, patients may suffer a relapse. It is important to identify such patients so that therapy tactics can be modified appropriately. Existing stratification schemes do not display the probability of recurrence with enough precision since locally advanced prostate cancer (PCa) is classified as high-risk but is not ranked in greater detail. Between 40 and 50% of PCa cases belong to the TMPRSS2-ERG subtype that is a sufficiently homogeneous group for high-precision prognostic marker search to be possible. This study includes two independent cohorts and is based on high throughput sequencing and qPCR data. As a result, we have been able to suggest a perspective-trained model involving a deep neural network based on both qPCR data for mRNA and miRNA and clinicopathological criteria that can be used for recurrence risk forecasts in patients with TMPRSS2-ERG-positive, locally advanced PCa (the model uses ALDH3A2 + ODF2 + QSOX2 + hsa-miR-503-5p + ISUP + pT, with an AUC = 0.944). In addition to the prognostic model's use of identified differentially expressed genes and miRNAs, miRNA-target pairs were found that correlate with the prognosis and can be presented as an interactome network.

Small RNA-Seq Reveals Similar miRNA Transcriptome in Children and Young Adults with T-ALL and Indicates miR-143-3p as Novel Candidate Tumor Suppressor in This Leukemia.

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.

Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes.

BACKGROUND: Exosomes are extracellular vesicles (EVs) derived from endocytic compartments of eukaryotic cells which contain various biomolecules like mRNAs or miRNAs. Exosomes influence the biologic behaviour and progression of malignancies and are promising candidates as non-invasive diagnostic biomarkers or as targets for therapeutic interventions. Usually, quantitative real-time polymerase chain reaction (qRT-PCR) is used to assess gene expression in cancer exosomes, however, the ideal reference genes for normalization yet remain to be identified. RESULTS: In this study, we performed an unbiased analysis of high-throughput mRNA and miRNA-sequencing data from exosomes of patients with various cancer types and identify candidate reference genes and miRNAs in cancer exosomes. The expression stability of these candidate reference genes was evaluated by the coefficient of variation "CV" and the average expression stability value "M". We subsequently validated these candidate reference genes in exosomes from an independent cohort of ovarian cancer patients and healthy control individuals by qRT-PCR. CONCLUSIONS: Our study identifies OAZ1 and hsa-miR-6835-3p as the most reliable individual reference genes for mRNA and miRNA quantification, respectively. For superior accuracy, we recommend the use of a combination of reference genes - OAZ1/SERF2/MPP1 for mRNA and hsa-miR-6835-3p/hsa-miR-4468-3p for miRNA analyses.

MiRNA-seq analysis of spleen and head kidney tissue from aquacultured largemouth bass (Micropterus salmoides) in response to Aeromonas hydrophila infection.

Recently, the same fish diseases, which have been found in pond farming, have been found in the newly tested largemouth bass (Micropterus salmoides) system. Bacterial septicemia caused by Aeromonas hydrophila occurs frequently in largemouth bass culture leading to significant economic losses. To investigate the role miRNA in the largemouth bass disease resistance, twelve (2 tissues (spleen and head kidney) × 2 experimental groups (infected and control) × three biological replicates) small RNA libraries were constructed and sequenced with miRNA-seq. A total of 26 differentially expressed miRNAs, 8 upregulated and 18 downregulated, were identified in the spleen, and 19 differentially expressed miRNAs, 9 upregulated and 10 downregulated, were identified in head kidney (fold change ≥ 2 or ≤ 0.5 and P ≤ 0.05). The differentially expressed miRNAs with the largest fold change were selected for target gene prediction using GO and KEGG analysis. Six miRNAs in the spleen and 5 miRNAs in the head kidney were selected. Analysis showed that, of all the immune and metabolic pathways, the FoxO signaling pathway was enriched in both the spleen and head kidney groups. Common target genes of the pathway included AMP-activated catalytic subunit alpha 1 (prkaa1), phosphatidylinositol 3-kinase (pik3r3b), serine/threonine-protein kinase (plk2), and forkhead box protein G1 (foxg1a). MiRNAs (such as miR-126-5P, miR-126-3P) are involved in immune response and cell cycle functions as they regulate targeted genes in the FoxO pathway. These results will enhance our understanding of the molecular mechanisms underlying immune responses to bacterial septicemia and facilitate molecular-assisted selection of resistant strains of largemouth bass.

Altered miRNA expression profiling in enamel organ of fluoride affected rat embryos.

Evidence has shown that miRNAs could play a role in dental fluorosis, but there is no study has investigated the global expression miRNA profiles of fluoride-exposed enamel organ. In this study, we analysed the differentially expressed (DE) miRNAs between fluoride-treated and control enamel organ for the first time and found several candidate miRNAs and signaling pathways worthy of further research. Thirty Wistar rats were randomly distributed into three groups and exposed to drinking water with different fluoride contents for 10 weeks and during the gestation. The three groups were a control group (distilled water), medium fluoride group (75 mg/L NaF), and high fluoride group (150 mg/L NaF). On the embryonic day 19.5, the mandible was dissected for histological analysis, and the enamel organ of the mandibular first molar tooth germ was collected for miRNA sequencing (miRNA-seq) and quantitative real-time PCR analysis (qRT-PCR). Typical dental fluorosis was observed in the incisors of the prepregnant rats. In addition to the disorganized structure of enamel organ cells, 39 DE miRNAs were identified in the fluoride groups compared with the control group, and good agreement between the miRNA-seq data and qRT-PCR data was found. The functional annotation of the target genes of 39 DE miRNAs showed significant enrichment in metabolic process, cell differentiation, calcium signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway terms. This study provides a theoretical reference for an extensive understanding of the mechanism of fluorosis and potential valuable miRNAs as therapeutic targets in fluorosis.

The Effects of Naringenin on miRNA-mRNA Profiles in HepaRG Cells.

Naringenin, a natural flavonoid widely found in citrus fruits, has been reported to possess anti-oxidant, anti-inflammatory, and hepatoprotective properties as a natural dietary supplement. However, the regulatory mechanism of naringenin in human liver remains unclear. In the present study, messenger RNA sequencing (mRNA-seq), microRNA sequencing (miRNA-seq), and real-time qPCR were used to distinguish the expression differences between control and naringenin-treated HepaRG cells. We obtained 1037 differentially expressed mRNAs and 234 miRNAs. According to the target prediction and integration analysis in silico, we found 20 potential miRNA-mRNA pairs involved in liver metabolism. This study is the first to provide a perspective of miRNA-mRNA interactions in the regulation of naringenin via an integrated analysis of mRNA-seq and miRNA-seq in HepaRG cells, which further characterizes the nutraceutical value of naringenin as a food additive.

High-throughput sequencing reveals crucial miRNAs in skeletal muscle development of Bian chicken.

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.

Chi-miR-30b-5p inhibits dermal papilla cells proliferation by targeting CaMKIIδ gene in cashmere goat.

BACKGROUND: During goat embryonic morphogenesis and postnatal initiation of hair follicle (HF) regeneration, dermal papilla (DP) cells play a vital role in hair formation. Growing evidence shows that microRNAs (miRNAs) participate in HF development and DP cell proliferation. However, the molecular mechanisms have not been thoroughly investigated. RESULT: In this study, we utilized miRNA sequencing (miRNA-Seq) to identify differentially expressed miRNAs at different HF cycling stages (anagen and telogen). MiRNA-Seq has identified 411 annotated miRNAs and 130 novel miRNAs in which 29 miRNAs were up-regulated and 32 miRNAs were down-regulated in the anagen phase compared to the telogen phase. Target gene prediction and functional enrichment analysis indicated some major biological pathways related to hair cycling, such as Wnt signaling pathways, ECM-receptor interaction, VEGF signaling pathway, biosynthesis of amino acids, metabolic pathways, ribosome and oxidative phosphorylation. Also, we explored the function of chi-miR-30b-5p in regulating hair growth cycle. Similar to the HF cycling, DP cells were isolated from skin and used to investigate miRNA functions. The MTT and EdU assays showed that the viability and proliferation of DP cells were inhibited or promoted after the transfection of chi-miR-30b-5p mimic or inhibitor, respectively. Bioinformatics analysis revealed CaMKIIδ as a candidate target gene of chi-miR-30b-5p, and the dual-luciferase and western blot assay demonstrated that chi-miR-30b-5p bound to the 3'UTR of CaMKIIδ and further inhibited its translation. CONCLUSION: Chi-miR-30b-5p was found to be highly expressed in the telogen than that in the anagen phase and could inhibit the proliferation of DP cells by targeting CaMKIIδ. Our study provides new information on the regulatory functions of miRNAs during HF development.