Humanized GPRC6AKGKY is a gain-of-function polymorphism in mice.

GPRC6A is proposed to regulate energy metabolism in mice, but in humans a KGKY polymorphism in the third intracellular loop (ICL3) is proposed to result in intracellular retention and loss-of-function. To test physiological importance of this human polymorphism in vivo, we performed targeted genomic humanization of mice by using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system to replace the RKLP sequence in the ICL3 of the GPRC6A mouse gene with the uniquely human KGKY sequence to create Gprc6a-KGKY-knockin mice. Knock-in of a human KGKY sequence resulted in a reduction in basal blood glucose levels and increased circulating serum insulin and FGF-21 concentrations. Gprc6a-KGKY-knockin mice demonstrated improved glucose tolerance, despite impaired insulin sensitivity and enhanced pyruvate-mediated gluconeogenesis. Liver transcriptome analysis of Gprc6a-KGKY-knockin mice identified alterations in glucose, glycogen and fat metabolism pathways. Thus, the uniquely human GPRC6A-KGKY variant appears to be a gain-of-function polymorphism that positively regulates energy metabolism in mice.

Role of GPRC6A in Regulating Hepatic Energy Metabolism in Mice.

GPRC6A is a widely expressed G-protein coupled receptor that regulates energy metabolism. Global deletion of Gprc6a in mice is reported to result in a metabolic syndrome-like phenotype and conditional deletion of Gprc6a in pancreatic ß-cell and skeletal muscle respectively impair insulin secretion and glucose uptake. In the current study, we explore the hepatic functions of GPRC6A by conditionally deleting Gprc6a in hepatocytes by cross breeding Alb-Cre and Gprc6aflox/flox mice to obtain Gprc6aLiver-cko mice. Gprc6aLiver-cko mice on a normal diet showed excessive hepatic fat accumulation and glycogen depletion. These mice also exhibit impaired glucose and pyruvate tolerance, but normal insulin sensitivity. Decreased circulating FGF-21 levels and FGF-21 message expression in the liver were found in Gprc6aLiver-cko mice. Hepatic transcriptome analysis identified alterations in multiple pathways regulating glucose, fat and glycogen metabolism in Gprc6aLiver-cko mice. Taken together, our studies suggest that GPRC6A directly regulates hepatic metabolism as well as regulates the production and release of FGF-21 to control systemic energy homeostasis. GPRC6A's unique regulation of ß-cell, skeletal muscle and hepatic function may represent a new therapeutic target for treating disordered energy metabolism metabolic syndrome and type 2 diabetes.

Plant MIR167e-5p Inhibits Enterocyte Proliferation by Targeting ß-Catenin.

MicroRNAs (miRNAs) are important negative regulators of genes involved in physiological and pathological processes in plants and animals. It is worth exploring whether plant miRNAs play a cross-kingdom regulatory role in animals. Herein, we found that plant MIR167e-5p regulates the proliferation of enterocytes in vitro. A porcine jejunum epithelial cell line (IPEC-J2) and a human colon carcinoma cell line (Caco-2) were treated with 0, 10, 20, and 40 pmol of synthetic 2'-O-methylated plant MIR167e-5p, followed by a treatment with 20 pmol of MIR167e-5p for 0, 24, 48, and 72 h. The cells were counted, and IPEC-J2 cell viability was determined by the MTT and EdU assays at different time points. The results showed that MIR167e-5p significantly inhibited the proliferation of enterocytes in a dose- and time-dependent manner. Bioinformatics prediction and a luciferase reporter assay indicated that MIR167e-5p targets ß-catenin. In IPEC-J2 and Caco-2 cells, MIR167e-5p suppressed proliferation by downregulating ß-catenin mRNA and protein levels. MIR167e-5p relieved this inhibition. Similar results were achieved for the ß-catenin downstream target gene c-Myc and the proliferation-associated gene PCNA. This research demonstrates that plant MIR167e-5p can inhibit enterocyte proliferation by targeting the ß-catenin pathway. More importantly, plant miRNAs may be a new class of bioactive molecules for epigenetic regulation in humans and animals.

Plant MIR156 regulates intestinal growth in mammals by targeting the Wnt/ß-catenin pathway.

MicroRNAs (miRNAs) are important negative regulators of genes involved in physiological and pathological processes in plants and animals. Recent studies have shown that miRNAs might regulate gene expression among different species in a cross-kingdom manner. However, the specific roles of plant miRNAs in animals remain poorly understood and somewhat. Herein, we found that plant MIR156 regulates proliferation of intestinal cells both in vitro and in vivo. Continuous administration of a high plant miRNA diet or synthetic MIR156 elevated MIR156 levels and inhibited the Wnt/ß-catenin signaling pathway in mouse intestine. Bioinformatics predictions and luciferase reporter assays indicated that MIR156 targets Wnt10b. In vitro, MIR156 suppressed proliferation by downregulating the Wnt10b protein and upregulating ß-catenin phosphorylation in the porcine jejunum epithelial (IPEC-J2) cell line. Lithium chloride and an MIR156 inhibitor relieved this inhibition. This research is the first to demonstrate that plant MIR156 inhibits intestinal cell proliferation by targeting Wnt10b. More importantly, plant miRNAs may represent a new class of bioactive molecules that act as epigenetic regulators in humans and other animals.

Human GPRC6A Mediates Testosterone-Induced Mitogen-Activated Protein Kinases and mTORC1 Signaling in Prostate Cancer Cells.

G protein-coupled receptor family C group 6 member A (GPRC6A) is activated by testosterone and modulates prostate cancer progression. Most humans have a GPRC6A variant that contains a recently evolved KGKY insertion/deletion in the third intracellular loop (ICL3) (designated as GPRC6AICL3_KGKY) that replaces the ancestral KGRKLP sequence (GPRC6AICL3_RKLP) present in all other species. In vitro assays purport that human GPRC6AICL3_KGKY is retained intracellularly and lacks function. These findings contrast with ligand-dependent activation and coupling to mammalian target of rapamycin complex 1 (mTORC1) signaling of endogenous human GPRC6AICL3_KGKY in PC-3 cells. To understand these discrepant results, we expressed mouse (mGPRC6AICL3_KGRKLP), human (hGPRC6AICL3_KGKY), and humanized mouse (mGPRC6AICL3_KGKY) GPRC6A into human embryonic kidney 293 cells. Our results demonstrate that mGPRC6AICL3_KGRKLP acts as a classic G protein-coupled receptor, which is expressed at the cell membrane and internalizes in response to ligand activation by testosterone. In contrast, hGPRC6AICL3_KGKY and humanized mouse mGPRC6AICL3_KGKY are retained intracellularly in ligand naive cells, yet exhibit ß-arrestin-dependent signaling responses, mitogen-activated protein kinase [i.e., extracellular signal-regulated kinase (ERK)], and p70S6 kinase phosphorylation in response to testosterone, indicating that hGPRC6AICL3_KGKY is functional. Indeed, testosterone stimulates time- and dose-dependent activation of ERK, protein kinase B, and mTORC1 signaling in wild-type PC-3 cells that express endogenous GPRC6AICL3_KGKY In addition, testosterone stimulates GPRC6A-dependent cell proliferation in wild-type PC-3 cells and inhibits autophagy by activating mTORC1 effectors eukaryotic translation initiation factor 4E binding protein 1 and Unc-51 like autophagy activating kinase 1. Testosterone activation of GPRC6A has the obligate requirement for calcium in the incubation media. In contrast, in GPRC6A-deficient cells, the effect of testosterone to activate downstream signaling is abolished, indicating that human GPRC6A is required for mediating the effects of testosterone on cell proliferation and autophagy.

Cardiovascular Interactions between Fibroblast Growth Factor-23 and Angiotensin II.

Both the activation of the renin angiotensin aldosterone system (RAAS) and elevations of circulating Fibroblast Growth Factor-23 (FGF-23) have been implicated in the pathogenesis of left ventricular hypertrophy (LVH) in chronic kidney disease. To investigate potential cross-talk between RAAS and FGF-23, we administered angiotensin II (Ang II) to wild-type rodents and the Hyp mouse model of excess FGF-23. Ang II administration for four weeks to wild-type rodents resulted in significant increases in systolic blood pressure and LVH. Unexpectedly, FGF-23 circulating levels were increased by 1.5-1.7 fold in Ang II treated animals. In addition, Ang II treatment increased expression of FGF-23 message levels in bone, the predominant tissue for FGF-23 production, and induced expression of FGF-23 and its co-receptor α-Klotho in the heart, which normally does not express FGF-23 or α-Klotho in physiologically relevant levels. Hyp mice with elevated FGF-23 exhibited increased blood pressure and LVH at baseline. Ang II administration to Hyp mice resulted further increments in blood pressure and left ventricular hypertrophy, consistent with additive cardiovascular effects. These findings suggest that FGF-23 may participate in unexpected systemic and paracrine networks regulating hemodynamic and myocardial responses.

Computationally identified novel agonists for GPRC6A.

New insights into G protein coupled receptor regulation of glucose metabolism by ß-cells, skeletal muscle and liver hepatocytes identify GPRC6A as a potential therapeutic target for treating type 2 diabetes mellitus (T2D). Activating GPRC6A with a small molecule drug represents a potential paradigm-shifting opportunity to make significant strides in regulating glucose homeostasis by simultaneously correcting multiple metabolic derangements that underlie T2D, including abnormalities in ß-cell proliferation and insulin secretion and peripheral insulin resistance. Using a computational, structure-based high-throughput screening approach, we identified novel tri-phenyl compounds predicted to bind to the venus fly trap (VFT) and 7-transmembrane (7-TM) domains of GPRC6A. Experimental testing found that these compounds dose-dependently stimulated GPRC6A signaling in a heterologous cell expression system. Additional chemical modifications and functional analysis identified one tri-phenyl lead compound, DJ-V-159 that demonstrated the greatest potency in stimulating insulin secretion in ß-cells and lowering serum glucose in wild-type mice. Collectively, these studies show that GPRC6A is a "druggable" target for developing chemical probes to treat T2DM.

GPCR6A Is a Molecular Target for the Natural Products Gallate and EGCG in Green Tea.

SCOPE: The molecular mechanisms whereby gallates in green tea exert metabolic effects are poorly understood. METHODS AND RESULTS: We found that GPRC6A, a multi-ligand-sensing G-protein-coupled receptor that regulates energy metabolism, sex hormone production, and prostate cancer progression, is a target for gallates. Sodium gallate (SG), gallic acid (GA) > ethyl gallate (EG) > octyl gallate (OG) dose dependently activated ERK in HEK-293 cells transfected with GPRC6A but not in non-transfected controls. SG also stimulated insulin secretion in ß-cells isolated from wild-type mice similar to the endogenous GPRC6A ligands, osteocalcin (Ocn) and testosterone (T). Side-chain additions to create OG resulted in loss of GPRC6A agonist activity. Another component of green tea, epigallocatechin 3-gallate (EGCG), dose-dependently inhibited Ocn activation of GPRC6A in HEK-293 cells transfected with GPRC6A and blocked the effect of Ocn in stimulating glucose production in CH10T1/2 cells. Using structural models of the venus fly trap (VFT) and 7-transmembrane (7-TM) domains of GPRC6A, calculations suggest that l-amino acids and GA bind to the VFT, whereas EGCG is calculated to bind to sites in both the VFT and 7-TM. CONCLUSION: GA and EGCG have offsetting agonist and antagonist effects on GPRC6A that may account for the variable metabolic effect of green tea consumption.

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.

CRISPR/Cas9 targeting of GPRC6A suppresses prostate cancer tumorigenesis in a human xenograft model.

BACKGROUND: GPRC6A is implicated in the pathogenesis of prostate cancer, but its role remains uncertain because of a purported tolerant gene variant created by substitution of a K..Y polymorphism in the 3rd intracellular loop (IL) that evolved in the majority of humans and replaces the ancestral RKLP present in 40% of humans of African descent and all other species. METHODS: We determined whether the K..Y polymorphism is present in human-derived prostate cancer cell lines by sequencing the region of the 3rd IL and assessed the cellular localization of a "humanized" mouse GPRC6A containing the K..Y sequence by immunofluorescence. We assessed functions of GPRC6A in PC-3 cells expressing endogenous GPRC6A and in GPRC6A-deficient PC-3 cells created using CRISPR/Cas9 technology. The effect of GPRC6A on basal and ligand stimulated cell proliferation and migration was evaluated in vitro in wild-type and PC-3-deficient cell lines. The effect of editing GPRC6A on prostate cancer growth and progression in vivo was assessed in a Xenograft mouse model implanted with wild-type and PC-3 deficient cells and treated with the GPRC6A ligand osteocalcin. RESULTS: We found that all of the human prostate cancer cell lines tested endogenously express the "K..Y" polymorphism in the 3rd IL. Comparison of mouse wild-type GPRC6A with a "humanized" mouse GPRC6A construct created by replacing the "RKLP" with the "K..Y" sequence, found that both receptors were predominantly expressed on the cell surface. The transfected "humanized" GPRC6A receptor, however, preferentially activated mTOR compared to ERK signaling in HEK-293 cells. In contrast, in PC-3 cells expressing the endogenous GPRC6A with the "K..Y" polymorphism, the ligand osteocalcin stimulated ERK, AKT and mTOR phosphorylation, promoted cell proliferation and migration, and upregulated genes regulating testosterone biosynthesis. Targeting GPRC6A in PC-3 cells by CRISPR/Cas9 significantly blocked these responses in vitro. In addition, GPRC6A deficient PC-3 xenografts exhibited significantly less growth and were resistant to osteocalcin-induced prostate cancer progression compared to control PC-3 cells expressing GPRC6A. CONCLUSIONS: Human GPRC6A is a functional osteocalcin and testosterone sensing receptor that promotes prostate cancer progression. GPRC6A may contribute to racial disparities in prostate cancer, and is a potential therapeutic target to develop antagonists to treat prostate cancer.

Revelation of mRNAs and proteins in porcine milk exosomes by transcriptomic and proteomic analysis.

BACKGROUND: Milk is a complex liquid that provides nutrition to newborns. Recent reports demonstrated that milk is enriched in maternal-derived exosomes that are involved in fetal physiological and pathological conditions by transmission of exosomal mRNAs, miRNAs and proteins. Until now, there is no such research relevant to exosomal mRNAs and proteins in porcine milk, therefore, we have attempted to investigate porcine milk exosomal mRNAs and proteins using RNA-sequencing and proteomic analysis. RESULTS: A total of 16,304 (13,895 known and 2,409 novel mRNAs) mRNAs and 639 (571 known, 66 candidate and 2 putative proteins) proteins were identified. GO and KEGG annotation indicated that most proteins were located in the cytoplasm and participated in many immunity and disease-related pathways, and some mRNAs were closely related to metabolisms, degradation and signaling pathways. Interestingly, 19 categories of proteins were tissue-specific and detected in placenta, liver, milk, plasma and mammary. COG analysis divided the identified mRNAs and proteins into 6 and 23 categories, respectively, 18 mRNAs and 10 proteins appeared to be involved in cell cycle control, cell division and chromosome partitioning. Additionally, 14 selected mRNAs were identified by qPCR, meanwhile, 10 proteins related to immunity and cell proliferation were detected by Western blot. CONCLUSIONS: These results provide the first insight into porcine milk exosomal mRNA and proteins, and will facilitate further research into the physiological significance of milk exosomes for infants.

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.

In low protein diets, microRNA-19b regulates urea synthesis by targeting SIRT5.

Ammonia detoxification, which takes place via the hepatic urea cycle, is essential for nitrogen homeostasis and physiological well-being. It has been reported that a reduction in dietary protein reduces urea nitrogen. MicroRNAs (miRNAs) are major regulatory non-coding RNAs that have significant effects on several metabolic pathways; however, little is known on whether miRNAs regulate hepatic urea synthesis. The objective of this study was to assess the miRNA expression profile in a low protein diet and identify miRNAs involved in the regulation of the hepatic urea cycle using a porcine model. Weaned 28-days old piglets were fed a corn-soybean normal protein diet (NP) or a corn-soybean low protein diet (LP) for 30 d. Hepatic and blood samples were collected, and the miRNA expression profile was assessed by sequencing and qRT-PCR. Furthermore, we evaluated the possible role of miR-19b in urea synthesis regulation. There were 25 differentially expressed miRNAs between the NP and LP groups. Six of these miRNAs were predicted to be involved in urea cycle metabolism. MiR-19b negatively regulated urea synthesis by targeting SIRT5, which is a positive regulator of CPS1, the rate limiting enzyme in the urea cycle. Our study presented a novel explanation of ureagenesis regulation by miRNAs.

Porcine milk-derived exosomes promote proliferation of intestinal epithelial cells.

Milk-derived exosomes were identified as a novel mechanism of mother-to-child transmission of regulatory molecules, but their functions in intestinal tissues of neonates are not well-studied. Here, we characterized potential roles of porcine milk-derived exosomes in the intestinal tract. In vitro, treatment with milk-derived exosomes (27 ± 3 ng and 55 ± 5 ng total RNA) significantly promoted IPEC-J2 cell proliferation by MTT, CCK8, EdU fluorescence and EdU flow cytometry assays. The qRT-PCR and Western blot analyses indicated milk-derived exosomes (0.27 ± 0.03 µg total RNA) significantly promoted expression of CDX2, IGF-1R and PCNA, and inhibited p53 gene expression involved in intestinal proliferation. Additionally, six detected miRNAs were significantly increased in IPEC-J2 cell, while FAS and SERPINE were significantly down-regulated relative to that in control. In vivo, treated groups (0.125 µg and 0.25 µg total RNA) significantly raised mice' villus height, crypt depth and ratio of villus length to crypt depth of intestinal tissues, significantly increased CDX2, PCNA and IGF-1R' expression and significantly inhibited p53' expression. Our study demonstrated that milk-derived exosomes can facilitate intestinal cell proliferation and intestinal tract development, thus giving a new insight for milk nutrition and newborn development and health.

Evidence for Osteocalcin Binding and Activation of GPRC6A in ß-Cells.

The possibility that G protein-coupled receptor family C member A (GPRC6A) is the osteocalcin (Ocn)-sensing G protein-coupled receptor that directly regulates pancreatic ß-cell functions is controversial. In the current study, we found that Ocn and an Ocn-derived C-terminal hexapeptide directly activate GPRC6A-dependent ERK signaling in vitro. Computational models probe the structural basis of Ocn binding to GPRC6A and predict that the C-terminal hexapeptide docks to the extracellular side of the transmembrane domain of GPRC6A. Consistent with the modeling, mutations in the computationally identified binding pocket of GPRC6A reduced Ocn and C-terminal hexapeptide activation of this receptor. In addition, selective deletion of Gprc6a in ß-cells (Gprc6a(ß)(-cell-cko)) by crossing Gprc6a(flox/flox) mice with Ins2-Cre mice resulted in reduced pancreatic weight, islet number, insulin protein content, and insulin message expression. Both islet size and ß-cell proliferation were reduced in Gprc6a(ß)(-cell-cko) compared with control mice. Gprc6a(ß)(-cell-cko) exhibited abnormal glucose tolerance, but normal insulin sensitivity. Islets isolated from Gprc6a(ß)(-cell-cko) mice showed reduced insulin simulation index in response to Ocn. These data establish the structural basis for Ocn direct activation of GPRC6A and confirm a role for GPRC6A in regulating ß-cell proliferation and insulin secretion.

Structural and Functional Evidence for Testosterone Activation of GPRC6A in Peripheral Tissues.

G protein-coupled receptor (GPCR) family C group 6 member A (GPRC6A) is a multiligand GPCR that is activated by cations, L-amino acids, and osteocalcin. GPRC6A plays an important role in the regulation of testosterone (T) production and energy metabolism in mice. T has rapid, transcription-independent (nongenomic) effects that are mediated by a putative GPCR. We previously found that T can activate GPRC6A in vitro, but the possibility that T is a ligand for GPRC6A remains controversial. Here, we demonstrate direct T binding to GPRC6A and construct computational structural models of GPRC6A that are used to identify potential binding poses of T. Mutations of the predicted binding site residues were experimentally found to block T activation of GPRC6A, in agreement with the modeling. Using Gpr6ca(-/-) mice, we confirmed that loss of GPRC6A resulted in loss of T rapid signaling responses and elucidated several biological functions regulated by GPRC6A-dependent T rapid signaling, including T stimulation of insulin secretion in pancreatic islets and enzyme expression involved in the biosynthesis of T in Leydig cells. Finally, we identified a stereo-specific effect of an R-isomer of a selective androgen receptor modulator that is predicted to bind to and shown to activate GPRC6A but not androgen receptor. Together, our data show that GPRC6A directly mediates the rapid signaling response to T and uncovers previously unrecognized endocrine networks.

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.

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.

Differentially expressed miRNAs after GnRH treatment and their potential roles in FSH regulation in porcine anterior pituitary cell.

Hypothalamic gonadotropin-releasing hormone (GnRH) is a major regulator of follicle-stimulating hormone (FSH) secretion in gonadotrope cell in the anterior pituitary gland. microRNAs (miRNAs) are small RNA molecules that control gene expression by imperfect binding to the 3'-untranslated region (3'-UTR) of mRNA at the post-transcriptional level. It has been proven that miRNAs play an important role in hormone response and/or regulation. However, little is known about miRNAs in the regulation of FSH secretion. In this study, primary anterior pituitary cells were treated with 100 nM GnRH. The supernatant of pituitary cell was collected for FSH determination by enzyme-linked immunosorbent assay (ELISA) at 3 hours and 6 hours post GnRH treatment respectively. Results revealed that GnRH significantly promoted FSH secretion at 3 h and 6 h post-treatment by 1.40-fold and 1.80-fold, respectively. FSHß mRNA at 6 h post GnRH treatment significantly increased by 1.60-fold. At 6 hours, cells were collected for miRNA expression profile analysis using MiRCURY LNA Array and quantitative PCR (qPCR). Consequently, 21 up-regulated and 10 down-regulated miRNAs were identified, and qPCR verification of 10 randomly selected miRNAs showed a strong correlation with microarray results. Chromosome location analysis indicated that 8 miRNAs were mapped to chromosome 12 and 4 miRNAs to chromosome X. Target and pathway analysis showed that some miRNAs may be associated with GnRH regulation pathways. In addition, In-depth analysis indicated that 10 up-regulated and 3 down-regulated miRNAs probably target FSHß mRNA 3'-UTR directly, including miR-361-3p, a highly conserved X-linked miRNA. Most importantly, functional experimental results showed that miR-361-3p was involved in FSH secretion regulation, and up-regulated miR-361-3p expression inhibited FSH secretion, while down-regulated miR-361-3p expression promoted FSH secretion in pig pituitary cell model. These differentially expressed miRNAs resolved in this study provide the first guide for post-transcriptional regulation of pituitary gonadotrope FSH secretion in pig, as well as in other mammals.