Bovine Milk Derived Exosomes Affect Gut Microbiota of DSS-Induced Colitis Mice.

The objective of this study was to investigate the effect of bovine milk derived exosomes (MDEs) on the gut microbiota of Dextran sodium sulfate (DSS)-induced colitis mice. Total of 42 specific pathogen free (SPF) male BALB/c mice (3 weeks old) were randomly assigned to three groups including control group, DSS group (DSS) and bovine milk derived exosome group (Exo), with 7 replicates/cages per treatment and two mice in one cage. 16S rRNA gene sequencing of cecal digesta samples was conducted. DSS significantly decreased the average daily feed intake of mice in DSS and Exo groups (P = 0.03). Shannon index of the DSS group was significantly lower than the control group (P < 0.05) whereas no difference between the control group and Exo group was observed. Administration of MDEs tended to increase the relative abundance of Campylobaterota. Compared to the control group, the relative abundance of Roseburia was significantly decreased in the DSS group (P < 0.05) whereas no difference between the Exo group and control group was observed. MDEs also tended to increase the relative abundance of Lachnospiraceae_UCG_006. In conclusion, oral administration of 10 µL MDEs (1 mg/mL) positively affected gut microbiota of DSS-induced colitis mice. The results of this study provided valuable reference for MDEs application in the prevention and treatment of colitis.

Protective effect of Enterococcus faecium against ethanol-induced gastric injury via extracellular vesicles.

Recently, Enterococcus has been shown to have gastric protective functions, and the mechanisms by which Enterococcus modulates gastric function are still being investigated. Herein, we investigated how Enterococcus faecium (Efm) and E. faecium-derived extracellular vesicles (EVs) (EfmEVs) exert protective effect against ethanol-induced gastric injury by investigating the effect of EfmEVs on gastric mucosal ulcer scoring, histological lesion, mucosal glycoprotein production, acidity, anti-oxidative function, and inflammatory responses in rat. Pretreatment with Efm showed significant reduction of ethanol-induced gastric injury, as evidenced by the lowering of ulcer index, histological lesion, gastric pH, and inflammatory responses and the enhancement of mucosal glycoprotein production and anti-oxidative function. Further functional studies on three bioactive components [inactivated Efm, EfmEVs (EVs), and EV-free supernatants] of the bacterial culture showed that EVs are mostly responsible for the gastroprotective effect. Moreover, EV secretion is beneficial for the gastroprotective effect of Efm. Hence, EVs mediated the protective effect of Efm against ethanol-induced gastric injury by lowering inflammatory responses and enhancing anti-oxidative function and may be a potent anti-inflammatory and anti-oxidative strategy to alleviate hyperinflammatory gastrointestinal tract conditions.IMPORTANCEThis study indicated that Enterococcus faecium provided a protective effect against rat gastric injury, which involved improvement of the mucosal glycoprotein production, anti-oxidative function, and inflammatory responses. Furthermore, we confirmed that three bioactive components (inactivated Efm, extracellular vesicles, and EV-free supernatants) of E. faecium culture also contributed to the gastroprotective effect. Importantly, E. faecium-derived EVs showed an effective impact for the gastroprotective effect.

Effects of Salmonella Outer Membrane Vesicles on Intestinal Microbiota and Intestinal Barrier Function.

Salmonella enterica is one of the most important zoonotic pathogens causing foodborne gastroenteritis worldwide. Outer membrane vesicles (OMVs) are lipid-bilayer vesicles produced by Gram-negative bacteria, which contain biologically active components. We hypothesized that OMVs are an important weapon of S. enterica to initiate enteric diseases pathologies. In this study, the effects of S. enterica OMVs (SeOMVs) on intestinal microbiota and intestinal barrier function were investigated. In vitro fecal culture experiments showed that alpha diversity indexes and microbiota composition were altered by SeOMV supplementation. SeOMV supplementation showed an increase of pH, a decrease of OD630 and total short chain fatty acid (SCFA) concentrations. In vitro IPEC-J2 cells culture experiments showed that SeOMV supplementation did not affect the IPEC-J2 cell viability and the indicated genes expression. In vivo experiments in mice showed that SeOMVs had adverse effects on average daily gain (p < 0.05) and feed:gain ratio (p < 0.05), and had a tendency to decrease the final body weight (p = 0.073) in mice. SeOMV administration decreased serum interleukin-10 level (p < 0.05), decreased the relative abundance of bacteria belonging to the genera BacC-u-018 and Akkermansia (p < 0.05). Furthermore, SeOMV administration damaged the ileum mucosa (p < 0.05). These findings suggest that SeOMVs play an important role in the activation of intestinal inflammatory response induced by S. enterica, and downregulation of SCFA-producing bacteria is a possible mechanism.

Effects of Dietary Essential Oils Supplementation on Egg Quality, Biochemical Parameters, and Gut Microbiota of Late-Laying Hens.

The objective of this study was to explore the effects of adding essential oils (EO) to diets on egg quality, biochemical parameters and intestinal flora of late laying hens. The number of 252 Dawu Golden Phoenix laying hens (55 weeks old) were randomly sorted into two groups: the control group (CG) fed a basal diet and the EO group fed a basal diet with 300 mg/kg of essential oils. The average egg weight, feed-to-egg ratio, and egg production rate were determined every week. The trial started at week 55 and lasted for 8 weeks. During the experiment's last week, 36 eggs out of each group were chosen at random to test. In our study, dietary supplementation with EO considerably decreased the egg breaking rate (p = 0.01) and increased the shell-breaking strength (p = 0.04). The treatment group's alanine aminotransferase (ALT) levels were considerably lower than those of the control group (p = 0.03). The EO group had substantially higher total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) (p = 0.04 and p =0.03, respectively). However, there were no differences in alpha diversity indicators between the two groups. It is worth noting that Firmicutes were increased considerably (p < 0.05), while Spirochaetota and Proteobacteria were significantly reduced in the EO group. At genus levels, the EO supplementation increased the relative abundance of Intestinimonas (p < 0.05) and Megamonas (p < 0.01). In conclusion, a dietary supplementation of 300 mg/kg EO can improve the production performance of laying hens and the egg quality. It can also regulate the abundance of cecal flora and serum biochemical indicators.

Effects of fulvic acid addition on laying performance, biochemical indices, and gut microbiota of aged hens.

The purpose of this study was to appraise the effect of fulvic acid on production, biochemical indices, and gut microbiota of laying hens. A total of 252 Dawu Golden Phoenix laying hens (55-week-old) were allotted to two treatments randomly, each with six replicates and 21 hens per replicate, including the control group (CG) and fulvic acid (500 mg/kg) group (FA). The trial period was 8 weeks. Adding FA raised egg weight (P = 0.03), shell-breaking strength (P = 0.03), and reduced egg breaking rate (P < 0.01), compared with CG. There was no difference in eggshell thickness and egg shape index between the two treatments; however, the FA group increased egg production by 1.45% and reduced the feed-to-egg ratio by 0.09. Moreover, dietary FA decreased the aspartate aminotransferase levels in serum (P = 0.04), and glutathione peroxidase and total antioxidant capacity were increased (P = 0.02 and 0.04, respectively). Despite this, the two groups had no differences in the alpha diversity indices (PD_whole trees, Shannon, Ace, Simpson, Chao1, and goods_coverage). Obviously, at the phylum level, the abundances of Firmicutes were improved (P < 0.01), Actinobacteriota (P < 0.01), and Proteobacteria (P < 0.01) were reduced by dietary FA. Supplementation with FA could improve the abundances of Megamonas (P < 0.01) and reduce Enterobacter (P < 0.01) at the genus level. To sum up, this study showed the addition of 500 mg/kg FA may boost production and egg quality and modulate the cecal microflora abundance and serum biochemical indices of laying hens.

Effects of Dietary Tributyrin on Growth Performance, Biochemical Indices, and Intestinal Microbiota of Yellow-Feathered Broilers.

This study aimed to evaluate the effects of tributyrin on growth performance, biochemical indices and intestinal microbiota of yellow-feathered broilers. 360 one-day-old chicks were randomly allocated to three treatments with six replicates of 20 chicks each, including a normal control group (NC), an antibiotic group (PC), and a tributyrin (250 mg/kg) group (TB) for 63 days. The results showed that compared with the control, the feed conversion ratio (FCR) in the TB group decreased during the d22 to d42 (p < 0.05) and overall, the final weight and FCR of broilers tended to increase and decrease, respectively. Moreover, the TB group showed the highest creatine concentrations at the entire period (p < 0.05). TB treatment increased the Bacteroidetes relative abundance and decreased Firmicutes. Principal coordinates analysis yielded clear clustering of the three groups. Linear discriminant analysis effect size analysis found seven differentially abundant taxa in the TB group, including several members of Bacteroidedetes. The relative abundance of Eisenbergiella, Phascolarctobacterium, Megasphaera and Intestinimonas increased in tributyrin-treated broilers. Spearman correlation analysis identified a correlation between Eisenbergiella abundance and overall feed efficiency. These results demonstrated that tributyrin could improve the growth performance by modulating blood biochemical indices and the cecal microflora composition of broilers.

Latest Trend of Milk Derived Exosomes: Cargos, Functions, and Applications.

Exosomes are nanosized phospholipid bilayer vesicles released to the extracellular environment. Exosomes from various tissues or cells are being studied and there has been a growing interest in milk exosomes research due to their emerging role as messengers between cells and the fact that it can be produced in large quantities with rich source of milk. Milk derived exosomes (MDEs) contain lipids, microRNAs, proteins, mRNAs as well as DNA. Studies of exosome cargo have been conducted widely in many research areas, especially exosomal miRNAs. In this paper, we reviewed the current knowledge in isolation and identification, cargos, functions mainly in intestinal tract and immunity system of MDEs. Its application as drug carriers and diseases biomarker are also discussed. Furthermore, we also consider critical challenges of MDEs application and provide possible directions for future research.

Research Note: Effects of glycerol monolaurate supplementation on egg production, biochemical indices, and gut microbiota of broiler breeders at the late stage of production.

The objective of the present study was to determine the effect of glycerol monolaurate (GML) supplementation on egg production, biochemical indices, and gut microbiota of broiler breeders at the late stage of production. Total of 180 healthy Qingyuan Partridge broiler breeders were randomly assigned to 2 groups: 1) corn-soybean meal based diet, and 2) basal diet supplemented with 300 mg glycerol monolaurate/kg. Each treatment group had 6 replicates with 15 birds within each replicate. The experiment started at wk 33 and lasted for 8 weeks. Feed conversion rate, egg weight, egg shape index, shell breaking strength, and shell thickness were not different between control and treatment groups. Supplementation of GML significantly decreased the egg breaking rate. All blood chemical indices and antioxidant parameters were not affected by GML except total antioxidant capacity which increased significantly with GML supplementation. Alpha diversity indices (Shannon, Simpson, Chao1, Ace, goods_coverage, and PD_whole tree) were not different between the 2 groups. Composition of cecal microbiota was not affected by GML supplementation except Euryarchaeota and Proteobacteria at phylum level. Overall, supplementation of glycerol monolaurate at 300 mg/kg level improved eggshell quality but its effect on cecal microbiota composition was limited on broiler breeders at the late stage of production.

Effects of monobutyrin supplementation on egg production, biochemical indexes, and gut microbiota of broiler breeders.

The objective of the present study was to determine the effect of monobutyrin supplementation on egg production, biochemical indexes, and gut microbiota of broiler breeders at the late stage of production. A total of 180 healthy Qingyuan partridge broilers were randomly assigned to 2 groups: 1) corn-soybean meal-based diet and 2) basal diet supplemented with 250 mg monobutyrin/kg. Each treatment group had 6 replicates/cages with 15 birds within each replicate. The experiment started at week 33 and lasted for 8 wk. Egg production rate, feed conversion rate, shell breaking strength, and shell thickness were not different between control and treatment groups. Supplementation of monobutyrin increased egg weight and tended to decrease egg breaking rate of Qingyuan partridge chickens. Supplementation of monobutyrin did not affect any of the biochemical indexes except total protein concentration. The 4 antioxidant parameters measured were not affected either. Alpha diversity indexes (Shannon, Simpson, Chao1, Ace, and Good's Coverage) and composition of cecal microbiota were not affected by monobutyrin supplementation. Overall, supplementation of monobutyrin at 250 mg/kg level improved egg quality, but its effect on cecal microbiota composition was limited.

Microbiome Analysis Investigating the Impacts of Fermented Spent Mushroom Substrates on the Composition of Microbiota in Weaned Piglets Hindgut.

The purpose of this study was to investigate the effects of fermented spent mushroom substrates (FSMS) on growth performance, serum biochemical, gut digestive enzyme activity, microbial community, genes expression of tight junction proteins, and volatile fatty acids in the hindgut (colon and cecum) of weaned piglets. A total of 100 weaned Yihao native pigs (native × Duroc, 50 males and 50 females) were allocated to two groups with five replicates and 10 pigs per replicate. Pigs in the control group were fed a basal diet (BD group), and the others were fed basal diets supplemented with 3% FSMS (FSMS group). Relative to the BD group, it had better results for final weight, average daily gain, and feed conversion ratio in the FSMS group but not significant (p > 0.05), which was accompanied by improved serum triiodothyronine, immunoglobulin G, and immunoglobulin A (p < 0.05) but lower serum total protein, albumin, total cholesterol, and total triglyceride during the overall period (p < 0.05). Similarly, FSMS significantly upregulated (p < 0.05) the messenger RNA expression of duodenal tight junction proteins such as tight junction protein 1, tight junction protein 2, and occludin. Meanwhile, isobutyric acid, valeric acid, and isovaleric acid levels were increased, whereas propanoic acid was decreased (p < 0.05) in the FSMS group than the BD group. In addition, the piglets in the FSMS group changed the microbial diversity in the colon and cecum. 16S rRNA gene sequencing-based compositional analysis of the colonic and cecal microbiota showed differences in the relative abundance of bacterial phyla (Firmicutes, Bacteroidetes, etc.), genus (Lactobacillus, Streptococcus, Roseburia, etc.), and species (Lactobacillus gasseri, Clostridium disporicum, etc.) between the BD and FSMS fed piglets. In conclusion, dietary supplementation with FSMS benefited to the intestinal mucosal barrier, immunity, and composition of the microbiota.

Pituitary miRNAs target GHRHR splice variants to regulate GH synthesis by mediating different intracellular signalling pathways.

Growth hormone (GH), whose synthesis and release are mainly regulated by intracellular signals mediated by growth hormone-releasing hormone receptor (GHRHR), is one of the major pituitary hormones and critical regulators of organism growth, metabolism, and immunoregulation. Pig GHRHR splice variants (SVs) may activate different signalling pathways via the variable C-terminal by alternative splicing, and SVs have the potential to change microRNA (miRNA) binding sites. In this study, we first confirmed the existence of pig GHRHR SVs (i.e., GHRHR, GHRHR SV1 and SV2) and demonstrated the inhibitory effects of critical pituitary miRNAs (i.e., let-7e and miR-328-5p) on GH synthesis and cell proliferation of primary pituitary cells. The SVs of GHRHR targeted by let-7e and miR-328-5p were predicted via bioinformatics analysis and verified by performing dual-luciferase reporter assays and detecting the expression of target transcripts. The differential responses of let-7e, and miR-328-5p to GH-releasing hormone and the changes in signalling pathways mediated by GHRHR suggested that let-7e and miR-328-5p were involved in GH synthesis mediated by GHRHR SVs, indicating that the two miRNAs played different roles by different ways. Finally, results showed that the protein coded by the GHRHR transcript regulated GH through the NO/NOS signalling pathway, whereas that coded by SV1 and SV2 regulated GH through the PKA/CREB signalling pathway, which was confirmed by the changes in signalling pathways after transfecting the expression vectors of GHRHR SVs to GH3 cells. To the best of our knowledge, this paper is the first to report pituitary miRNAs regulate GH synthesis by targeting the different SVs of GHRHR.

miR-709 inhibits GHRP6 induced GH synthesis by targeting PRKCA in pituitary.

Pituitary growth hormone (GH) plays an essential role in processes of organism growth and metabolism. MicroRNA (miRNA) could also participate in diverse biological processes. However, the role of miRNA in the regulation of pituitary GH during the growth process remains unclear. In this study, we firstly confirmed that the second highly expressed pituitary miRNA (miR-709) significantly inhibited the GH synthesis and suppressed the viability of GH3 cells. The bioinformatics analysis and dual luciferase report system were used to ascertain the PRKCA is the direct target gene of miR-709, which is the coding gene of PKCα. Then the transcription and translation levels of Prkca were obvious reduced by the over-expression of miR-709 in GH3 cells, followed by the inhibition of the transcription factor (CREB1) of Gh1 gene and the ERK1/2 signaling pathway or the possible cross-talk signaling pathway (cAMP/PKA signaling pathway) detected by western blot, suggesting that ERK1/2 maybe an important factor involved in the GH3 cell viability mediated by PKCα. At last, GHRP6 increased PKCα and GH expression but reduced miR-709 expression in vitro and vivo assays, and this conclusion was further confirmed by the result of GHRP6 attenuated the inhibition of miR-709 on GH expression. These findings will provide new molecular mechanism on the regulation of pituitary GH.

Age-Related Changes in MicroRNA in the Rat Pituitary and Potential Role in GH Regulation.

The growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis has recently been recognized as an important factor related to the longevity of many organisms. MicroRNAs (miRNAs or miRs) could also participate in diverse biological processes. However, the role of miRNAs in the decline of pituitary GH during the growth process remains unclear. To better characterize the effects of miRNAs on the pituitary, we used a miRNA microarray to investigate the miRNA profile in the rat pituitary from postnatal development throughout the growth process. Then, in vitro experiments were conducted to analyze the miRNAs' potential roles related to GH regulation. Taken together, the microarray results indicated that there were 22 miRNAs differentially expressed during pituitary development. The bioinformatics analysis suggested that the most differentially expressed miRNAs may participate in multiple pathways associated with the pituitary function. Furthermore, the in vitro findings demonstrated that miR-141-3p was involved in GH regulation.

miRNAome, mRNAome and degradome analysis of Tibetan minipigs anterior pituitary.

Tibetan minipig is an important animal model for human diseases. The anterior pituitary is the master gland responsible for growth, reproduction, and metabolism and is regulated by thousands of miRNAs/mRNAs molecules. However, little is known about miRNAs and their relationships with mRNAs in Tibetan minipig anterior pituitary. Using microarray and mRNA-Sequencing, we identified 203 miRNAs and 12,040 mRNA transcripts from the anterior pituitary of Tibetan minipigs. These miRNAs were corresponding to 194 hairpin precursors, 25 miRNA clusters and 24 miRNA families. In addition, 64 intragenic miRNAs were annotated. Using three bioinformatic algorithms (TargetScan, miRanda and RNAhybrid), 359,184 possible miRNA-mRNA interactions were predicted, and an integrated network of miRNAs and pituitary-specific mRNA transcripts was established. To validate the predicted results, the degradome sequencing was employed to confirm miRNA-mRNA interactions, totally, 30 miRNA-mRNA pairs were identified. The present study provided a general overview of miRNA and mRNA annotation in Tibetan minipig anterior pituitary and established a miRNA-mRNA interactions database at the whole genome scale, which helps shed light on the molecular mechanisms in the anterior pituitary of pigs even other mammals.

miR-361-3p regulates FSH by targeting FSHB in a porcine anterior pituitary cell model.

FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.

Comparative Anterior Pituitary miRNA and mRNA Expression Profiles of Bama Minipigs and Landrace Pigs Reveal Potential Molecular Network Involved in Animal Postnatal Growth.

The anterior pituitary is the most important endocrine organ modulating animal postnatal growth, mainly by controlling growth hormone (GH) gene transcription, synthesis, and secretion. As an ideal model for animal postnatal growth studies, the Bama minipig is characterized as having a lower growth performance and fewer individual differences compared with larger pig breeds. In this study, anterior pituitaries from Bama minipig and Landrace pig were used for miRNA and mRNA expression profile analysis using miRNA microarrays and mRNA-seq. Consequently, a total of 222 miRNAs and 12,909 transcripts were detected, and both miRNAs and mRNAs in the two breeds showed high correlation (r > 0.97). Additionally, 41 differentially expressed miRNAs and 2,254 transcripts were identified. Pathways analysis indicated that 32 pathways significantly differed in the two breeds. Importantly, two GH-regulation-signalling pathways, cAMP and inositol 1, 4, 5-triphosphate (IP3), and multiple GH-secretion-related transcripts were significantly down-regulated in Bama minipigs. Moreover, TargetScan and RNAHybrid algorithms were used for predicting differentially expressed miRNAs (DE miRNAs) and differentially expressed mRNAs (DE mRNAs) interaction. By examining their fold-changes, interestingly, most DE miRNA-DE mRNA target pairs (63.68-71.33%) presented negatively correlated expression pattern. A possible network among miRNAs, mRNAs, and GH-regulation pathways was also proposed. Among them, two miRNA-mRNA interactions (Y-47 targets FSHB; ssc-miR-133a-3p targets GNAI3) were validated by dual-luciferase assay. These data will be helpful in understanding the possible molecular mechanisms involved in animal postnatal growth.

Alteration of the miRNA expression profile in male porcine anterior pituitary cells in response to GHRH and CST and analysis of the potential roles for miRNAs in regulating GH.

OBJECTIVE: Growth hormone releasing hormone (GHRH) is a major positive regulator of growth hormone (GH) in the anterior pituitary gland, while cortistatin's (CST) role is negative. miRNAs (microRNAs or miRs) are small RNA molecules modulating gene expression at the post-transcriptional level. However, little is known about the function of miRNAs in the regulation of GH synthesis and/or secretion. This study investigated potential functional miRNAs involved in GH secretion in the normal porcine pituitary. DESIGN: Primary porcine anterior pituitary cells were cultivated and then treated with 10 nmol/L GHRH and 100 nmol/L CST, respectively. The effects of GHRH and CST on GH secretion were determined using RIA. miRNA microarrays were employed to analyze miRNA expression after treatment and then differentially expressed miRNAs were screened. Bioinformatics analysis was used to analyze the potential targets in growth hormone regulation of altered miRNAs. Furthermore, functional experiments were conducted to study the function of ssc-let-7c. RESULTS: GHRH significantly promoted GH secretion, while CST suppressed GH secretion. 19 and 35 differentially expressed miRNAs were identified in response to GHRH and CST treatments respectively. Verification of 5 randomly selected miRNAs by quantitative real-time PCR (qRT-PCR) showed similar changes with microarray analysis. Target analysis showed that some miRNAs may be involved in GH secretion-related pathways. Importantly, ssc-let-7c was predicted to target GH1 and GHRHR mRNA 3'untranslated regions (3'UTRs), which was supported by luciferase reporter assay. Furthermore, functional experimental results showed that ssc-let-7c was involved in GH secretion regulation, and overexpression of ssc-let-7c inhibited GH secretion in porcine anterior pituitary cells. CONCLUSIONS: GHRH and CST modulated porcine pituitary cell miRNA expression. Bioinformatics analysis revealed a complicated network among differentially expressed miRNAs, GH regulation-related genes and hormones. More interestingly, ssc-let-7c inhibited both GH1 and GHRHR mRNA 3'UTR reporter vectors' luciferase activity and overexpression of ssc-let-7c led to a decrease of GH secretion.

Genome-wide discovery of novel and conserved microRNAs in white shrimp (Litopenaeus vannamei).

Of late years, a large amount of conserved and species-specific microRNAs (miRNAs) have been performed on identification from species which are economically important but lack a full genome sequence. In this study, Solexa deep sequencing and cross-species miRNA microarray were used to detect miRNAs in white shrimp. We identified 239 conserved miRNAs, 14 miRNA* sequences and 20 novel miRNAs by bioinformatics analysis from 7,561,406 high-quality reads representing 325,370 distinct sequences. The all 20 novel miRNAs were species-specific in white shrimp and not homologous in other species. Using the conserved miRNAs from the miRBase database as a query set to search for homologs from shrimp expressed sequence tags (ESTs), 32 conserved computationally predicted miRNAs were discovered in shrimp. In addition, using microarray analysis in the shrimp fed with Panax ginseng polysaccharide complex, 151 conserved miRNAs were identified, 18 of which were significant up-expression, while 49 miRNAs were significant down-expression. In particular, qRT-PCR analysis was also performed for nine miRNAs in three shrimp tissues such as muscle, gill and hepatopancreas. Results showed that these miRNAs expression are tissue specific. Combining results of the three methods, we detected 20 novel and 394 conserved miRNAs. Verification with quantitative reverse transcription (qRT-PCR) and Northern blot showed a high confidentiality of data. The study provides the first comprehensive specific miRNA profile of white shrimp, which includes useful information for future investigations into the function of miRNAs in regulation of shrimp development and immunology.

Exploration of microRNAs in porcine milk exosomes.

BACKGROUND: Breast milk contains complex nutrients and facilitates the maturation of various biological systems in infants. Exosomes, membranous vesicles of endocytic origin found in different body fluids such as milk, can mediate intercellular communication. We hypothesized that microRNAs (miRNAs), a class of non-coding small RNAs of 18-25 nt which are known to be packaged in exosomes of human, bovine and porcine milk, may play important roles in the development of piglets. RESULTS: In this study, exosomes of approximately 100 nm in diameter were isolated from porcine milk through serial centrifugation and ultracentrifugation procedures. Total RNA was extracted from exosomes, and 5S ribosomal RNA was found to be the major RNA component. Solexa sequencing showed a total of 491 miRNAs, including 176 known miRNAs and 315 novel mature miRNAs (representing 366 pre-miRNAs), which were distributed among 30 clusters and 35 families, and two predicted novel miRNAs were verified targeting 3'UTR of IGF-1R by luciferase assay. Interestingly, we observed that three miRNAs (ssc-let-7e, ssc-miR-27a, and ssc-miR-30a) could be generated from miRNA-offset RNAs (moRNAs). The top 10 miRNAs accounted for 74.5% (67,154 counts) of total counts, which were predicted to target 2,333 genes by RNAhybrid software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using DAVID bioinformatics resources indicated that the identified miRNAs targeted genes enriched in transcription, immunity and metabolism processes, and 14 of the top 20 miRNAs possibly participate in regulation of the IgA immune network. CONCLUSIONS: Our findings suggest that porcine milk exosomes contain a large number of miRNAs, which potentially play an important role in information transfer from sow milk to piglets. The predicted miRNAs of porcine milk exosomes in this study provide a basis for future biochemical and biophysical function studies.

MiRNA-181a regulates adipogenesis by targeting tumor necrosis factor-α (TNF-α) in the porcine model.

Adipogenesis is tightly regulated by altering gene expression, and TNF-α is a multifunctional cytokine that plays an important role in regulating lipogenesis. MicroRNAs are strong post-transcriptional regulators of cell differentiation. In our previous work, we found high expression of miR-181a in a fat-rich pig breed. Using bioinformatic analysis, miR-181a was identified as a potential regulator of TNF-α. Here, we validated TNF-α as the target of miR-181a by a dual luciferase assay. In response to adipogenesis, a mimic or inhibitor was used to overexpress or reduce miR-181a expression in porcine pre-adipocytes, which were then induced into mature adipocytes. Overexpression of miR-181a accelerated accumulation of lipid droplets, increased the amount of triglycerides, and repressed TNF-α protein expression, while the inhibitor had the opposite effect. At the same time, TNF-alpha rescued the increased lipogenesis by miR181a mimics. Additionally, miR-181a suppression decreased the expression of fatty synthesis associated genes PDE3B (phosphodiesterase 3B), LPL (lipoprotein lipase), PPARγ (proliferator-activated receptor-γ), GLUT1 (glucose transporter), GLUT4, adiponectin and FASN (fatty acid synthase), as well as key lipolytic genes HSL (hormone-sensitive lipase) and ATGL (adipose triglyceride lipase) as revealed by quantitative real-time PCR. Our study provides the first evidence of the role of miR-181a in adipocyte differentiation by regulation of TNF-α, which may became a new therapeutic target for anti-obesity drugs.