MicroRNA expression and oxidative stress markers in pectoral muscle of broiler chickens fed diets supplemented with phytobiotics composition.

Phytobiotic compositions are commercially used in broiler production, mostly to improve general health and the production parameters. Moreover, some of their active substances may change the expression of miRNA in different tissues. Therefore, the purpose of this study was to evaluate the effect of the phytobiotic composition (PBC) containing white mustard, calamus, turmeric, and common ivy on production parameters, oxidative stress markers and expression of selected miRNAs in pectoral muscle of broiler chickens. The experiment was performed on broiler chickens fed the control diet (without PBC), and a diet supplemented with 60 or 100 mg/kg of PBC for 35 days. After the experiment, samples (blood and muscle) were collected for analyses. The analyzed production parameters included: feed conversion ratio, feed intake and body weight. There was no effect on growth performance of broiler chickens but feeding diet supplemented with 60 mg/kg phytobiotics significantly increased the expression of miR-30a-5p, miR-181a-5p, and miR-206, and decreased that of miR-99a-5p, miR-133a-5p, miR-142-5p, and miR-222 in pectoral muscle of chickens. The addition of 100 mg/kg phytobiotics significantly increased miR-99a-5p and miR-181a-5p expression, and caused down-regulation of the expression of miR-26a-5p and miR-30a-5p. Chickens fed diet supplemented with 100 mg/kg PBC had lower level of lipid peroxidation products in blood, while in the muscle tissue it was higher in birds fed a diet with the addition of 60 mg/kg as compared to the control group. The results suggest that this unique composition of phytobiotics does not affect productive traits but can change expression of miRNAs that are crucial for muscle physiology and pathology in broiler chickens. This additive may also protect against the oxidative stress but the effect is dose dependent.

Role of microRNAs and their putative mechanism in regulating potato (Solanum tuberosum L.) life cycle and response to various environmental stresses.

The exponentially increasing population and the demand for food is inextricably linked. This has shifted global attention to improving crop plant traits to meet global food demands. Potato (Solanum tuberosum L.) is a major non-grain food crop that is grown all over the world. Currently, some of the major global potato research work focuses on the significance of microRNAs (miRNAs) in potato. miRNAs are a type of non-coding RNAs that regulate the gene expression of their target mRNA genes by cleavage and/or their translational inhibition. This suggests an essential role of miRNAs in a multitude of plant biological processes, including maintenance of genome integrity, plant growth, development and maturation, and initiation of responses to various stress conditions. Therefore, engineering miRNAs to generate stress-resistant varieties of potato may result in high yield and improved nutritional qualities. In this review, we discuss the potato miRNAs specifically known to play an essential role in the various stages of the potato life cycle, conferring stress-resistant characteristics, and modifying gene expression. This review highlights the significance of the miRNA machinery in plants, especially potato, encouraging further research into engineering miRNAs to boost crop yields and tolerance towards stress.

FaesAP3_1 Regulates the FaesELF3 Gene Involved in Filament-Length Determination of Long-Homostyle Fagopyrum esculentum.

The identification downstream genes of floral organ identity regulators are critical to revealing the molecular mechanisms underlying floral morphogenesis. However, a general regulatory pathway between floral organ identity genes and their downstream targets is still unclear because of the lack of studies in nonmodel species. Here, we screened a direct downstream target gene, FaesELF3, of a stamen identity transcription factor, FaesAP3_1, in long-homostyle (LH) Fagopyrum esculentum moench by using yeast one-hybrid (Y1H) and dual-luciferase reporter (DR) assays. Furthermore, FaesAP3_1-silenced LH plants that produced flowers with part stamens or anthers homeotically converted into a tepaloid structure, and FaesELF3-silenced plants that had flowers with part stamens consisting of a short filament and empty anther (male sterile anther). All these suggested that transcription factor (TF) FaesAP3_1 directly activates FaesELF3 in order to regulate filament elongation and pollen grain development in LH buckwheat. Our data also suggested that other stamen development pathways independent of FaesAP3_1 remain in F. esculentum.

Screening of differentially expressed microRNAs and target genes in two potato varieties under nitrogen stress.

BACKGROUND: A reasonable supply of nitrogen (N) fertilizer is essential for obtaining high-quality, high-level, and stable potato yields, and an improvement in the N utilization efficiency can effectively reduce N fertilizer use. It is important to use accurate, straightforward, and efficient transgenic breeding techniques for the identification of genes that can improve nitrogen use efficiency, thus enabling us to achieve the ultimate goal of breeding N-efficient potato varieties. In recent years, some of the mechanisms of miRNAs have been elucidated via the analysis of the correlation between the expression levels of potato miRNA target genes and regulated genes under conditions of stress, but the role of miRNAs in the inhibition/expression of key genes regulating N metabolism under N stress is still unclear. Our study aimed to identify the role played by specific enzymes and miRNAs in the responses of plants to N stress. RESULTS: The roots and leaves of the N-efficient potato variety, Yanshu4 ("Y"), and N-inefficient potato variety, Atlantic ("D"), were collected at the seedling and budding stages after they were exposed to different N fertilizer treatments. The miRNAs expressed differentially under the two types of N stress and their corresponding target genes were first predicted using miRNA and degradome analysis. Then, quantitative polymerase chain reaction (qRT-PCR) was performed to verify the expression of differential miRNAs that were closely related to N metabolism. Finally, the shearing relationship between stu-miR396-5p and its target gene StNiR was determined by analyzing luciferase activity levels. The results showed that NiR activity increased significantly with an increase in the applied N levels from the seedling stage to the budding stage, and NiR responded significantly to different N treatments. miRNA sequencing enabled us to predict 48 families with conserved miRNAs that were mainly involved in N metabolism, carbon metabolism, and amino acid biosynthesis. The differences in the expression of the following miRNAs were identified via screening (high expression levels and P < 0.05): stu-miR396-5p, stu-miR408b-3p_R-1, stu-miR3627-3p, stu-miR482a-3p, stu-miR8036-3p, stu-miR482a-5p, stu-miR827-5p, stu-miR156a_L-1, stu-miR827-3p, stu-miR172b-5p, stu-miR6022-p3_7, stu-miR398a-5p, and stu-miR166c-5p_L-3. Degradome analysis showed that most miRNAs had many-to-many relationships with target genes. The main target genes involved in N metabolism were NiR, NiR1, NRT2.5, and NRT2.7. qRT-PCR analysis showed that there were significant differences in the expression levels of stu-miR396-5p, stu-miR8036-3p, and stu-miR482a-3p in the leaves and roots of the Yanshu4 and Atlantic varieties at the seedling and budding stages under conditions that involved no N and excessive N application; the expression of these miRNAs was induced in response to N stress. The correlation between the differential expression of stu-miR396-5p and its corresponding target gene NiR was further verified by determining the luciferase activity level and was found to be strongly negative. CONCLUSION: The activity of NiR was significantly positively correlated with N application from the seedling to the budding stage. Differential miRNAs and target genes showed a many-to-many relationship with each other. The expression of stu-miR396-5p, stu-miR482a-3p, and stu-miR8036-3p in the roots and leaves of the Yanshu4 and Atlantic varieties at the seedling and budding stages was notably different under two types of N stress. Under two types of N stress, stu-miR396-5p was down-regulated in Yanshu4 in the seedling-stage and shoot-stage roots, and up-regulated in seedling-stage roots and shoot-stage leaves; stu-miR482a-3p was up-regulated in the seedling and shoot stages. The expression of stu-miR8036-3p was up-regulated in the leaves and roots at the seedling and budding stages, and down-regulated in roots under both types of N stress. The gene expressing the key enzyme involved in N metabolism, StNiR, and the stu-miR396-5p luciferase assay reporter gene had a strong regulatory relationship with each other. This study provides candidate miRNAs related to nitrogen metabolism and highlights that differential miRNAs play a key role in nitrogen stress in potato, providing insights for future research on miRNAs and their target genes in nitrogen metabolic pathways and breeding nitrogen-efficient potatoes.

Genome-Wide Analysis of miR159 Gene Family and Predicted Target Genes Associated with Environmental Stress in Dendrobium officinale: A Bioinformatics Study.

Dendrobium officinale (D. officinale) is a widely used traditional Chinese medicine with high economic value. MicroR159 (miR159) is an ancient and conserved microRNA (miRNA) family in land plants, playing roles in the progress of growth and development, as well as the stress response. In order to find out functions of miR159 in D. officinale, multiple bioinformatic approaches were employed and 10 MIR159 genes were found, localizing on seven chromosomes and an unanchored segment of the D. officinale genome. All of the precursor sequences of Dof-miR159 could form a stable stem-loop structure. The phylogenetic analysis revealed that the MIR159 genes of D. officinale were divided into five clades. Furthermore, the conservation analysis suggested that the 2 to 20 nt region of miR159 mature sequences were highly conserved among family members. The promoter analysis of MIR159s showed that the majority of the predicted cis-elements were related to environmental stress or hormones. In total, five classes of genes were predicted to be the target genes of Dof-miR159s, including GAMYB transcription factors, which had been confirmed in many other land plants. The expression patterns of predicted target genes revealed their potential roles in the growth and development of D. officinale, as well as in cold and drought stress responses. In conclusion, our results illustrated the stress-related miR159-targeted genes in D. officinale, which could provide candidate genes for resistance breeding in the future.

[Identification of miR160 family and their target genes in Rehmannia glutinosa in response to endophytic fungus GG22 infection].

This study aims to reveal the possible role of miR160 family in Rehmannia glutinosa in response to the infection of endophytic fungus Fusarium oxysporum GG22. Specifically, miR160 precursors and mature miR160 were retrieved from the small RNA database yielded by high-throughput sequencing. RNAfold was used to analyze the precursor structure, and DNAMAN and MEGA to analyze conservation and evolution of miR160 precursors and mature miR160. The target genes of miR160 were predicted and annotated, and the interaction was analyzed. Based on degradome sequencing, the target genes were further identified. The results showed that miR160 precursors had intact stem-loop structures. The precursor and mature sequences were conserved, particularly the 3 rd-16 th bases of the 5'-terminal. According to the phylogenetic tree, R. glutinosa had close evolutionary relationship with Arabidopsis thaliana, Oryza sativa, Salvia miltiorrhiza, and Sesamum indicum. A total of 22 target genes of miR160 were predicted and most of them were auxin response factor(ARF) genes. The target genes were involved in the Gene Ontology(GO) terms of biological processes, cellular components, and molecular functions. According to the degradome sequencing results, four target genes of miR160 were ARF(ARF18, ARF22) genes. R. glutinosa regulated its growth in response to the infection of endophytic fungus by changing the expression of miR160 and the target genes. qRT-PCR result of the differentially expressed rgl-miR160a and rgl-miR160a-3p was consistent with the sequencing result. This study clarifies the molecular mechanism of R. glutinosa in response to GG22 stress, laying a theoretical basis for the improvement and future research of R. glutinosa.

miRNA and miRNA target genes in intervention effect of Zhuyu pill on cholestatic rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhuyu pill (ZYP), an effective prescription of traditional Chinese medicine, is composed of Coptis chinensis Franch. and Tetradium ruticarpum (A. Jussieu) T. G. Hartley and has shown potential anticholestatic effects. However, its mechanism of action in treating cholestasis remains unclear. Since post-transcriptional control of mRNA by micro-RNAs (miRNAs) represents an important mechanism of gene regulation, it is promising to explore this in relation to ZYP and cholestasis. AIM OF THE STUDY: To confirm the anticholestatic effect of ZYP and to explore its potential biological mechanism. MATERIALS AND METHODS: In this study, a cholestasis rat model was induced by α-naphthyl-isothiocyanate (ANIT, 50 mg/kg) and treated with ZYP (low dose: 0.6 g/kg, high dose: 1.2 g/kg). Serum biochemistry indices and liver histopathology were used to evaluate the model and efficacy, and miRNA sequencing was used to measure differences in miRNA expression in the liver between the control, model, low-dose ZYP, and high-dose ZYP groups. To verify the accuracy of sequencing results and explore the potential anti-cholestasis mechanism of ZYP, RT-PCR was used to identify differentially expressed miRNAs and their target genes. RESULTS: Both high- and low-dose ZYP exhibited significant anticholestatic effects, with the high-dose showing better effects than low-dose ZYP. Additionally, four differentially expressed miRNAs, rno-miR-147, rno-miR-20b-5p, rno-miR-29b-3p, and rno-miR-3586-3p, were found to be upregulated in cholestasis and downregulated after ZYP intervention. Eight target genes of the above miRNAs, including ABCG8, CLOCK, PLEC, SLC4A2, NEB, ADAMTS12, TTN and FAM174B were inhibited in cholestatic rats, exhibiting up-regulated expression tendencies after ZYP intervention, and the expression tendencies were significant negatively correlated with serum biochemical indices. CONCLUSIONS: ZYP can significantly reduce liver biochemical indices and improve liver tissue damage in cholestasis rats through the regulation of miRNA expression in the liver, producing a positive regulatory effect on bile excretion-related genes.

Identification and characterization of miRNAs associated with sterile flower buds in the tea plant based on small RNA sequencing.

BACKGROUND: miRNAs are a type of conserved, small RNA molecule that regulate gene expression and play an important role in the growth and development of plants. miRNAs are involved in seed germination, root development, shoot apical meristem maintenance, leaf development, and flower development by regulating various target genes. However, the role of miRNAs in the mechanism of tea plant flower sterility remains unclear. Therefore, we performed miRNA sequencing on the flowers of fertile male parents, female parents, and sterile offspring. RESULTS: A total of 55 known miRNAs and 90 unknown miRNAs were identified. In the infertile progeny, 37 miRNAs were differentially expressed; 18 were up-regulated and 19 were down-regulated. miR156, miR157, miR164, miR167, miR169, miR2111 and miR396 family members were down-regulated, and miR160, miR172 and miR319 family members were up-regulated. Moreover, we predicted that the 37 differentially expressed miRNAs target a total of 363 genes, which were enriched in 31 biological functions. We predicted that miR156 targets 142 genes, including ATD1A, SPL, ACA1, ACA2, CKB22 and MADS2. CONCLUSION: We detected a large number of differentially expressed miRNAs in the sterile tea plant flowers, and their target genes were involved in complex biological processes. Among these miRNAs, the down-regulation of miR156 may be one of the factor in the formation of sterile floral buds in tea plants.

Investigation of candidate genes and mechanisms underlying obesity associated type 2 diabetes mellitus using bioinformatics analysis and screening of small drug molecules.

BACKGROUND: Obesity associated type 2 diabetes mellitus is a metabolic disorder ; however, the etiology of obesity associated type 2 diabetes mellitus remains largely unknown. There is an urgent need to further broaden the understanding of the molecular mechanism associated in obesity associated type 2 diabetes mellitus. METHODS: To screen the differentially expressed genes (DEGs) that might play essential roles in obesity associated type 2 diabetes mellitus, the publicly available expression profiling by high throughput sequencing data (GSE143319) was downloaded and screened for DEGs. Then, Gene Ontology (GO) and REACTOME pathway enrichment analysis were performed. The protein - protein interaction network, miRNA - target genes regulatory network and TF-target gene regulatory network were constructed and analyzed for identification of hub and target genes. The hub genes were validated by receiver operating characteristic (ROC) curve analysis and RT- PCR analysis. Finally, a molecular docking study was performed on over expressed proteins to predict the target small drug molecules. RESULTS: A total of 820 DEGs were identified between healthy obese and metabolically unhealthy obese, among 409 up regulated and 411 down regulated genes. The GO enrichment analysis results showed that these DEGs were significantly enriched in ion transmembrane transport, intrinsic component of plasma membrane, transferase activity, transferring phosphorus-containing groups, cell adhesion, integral component of plasma membrane and signaling receptor binding, whereas, the REACTOME pathway enrichment analysis results showed that these DEGs were significantly enriched in integration of energy metabolism and extracellular matrix organization. The hub genes CEBPD, TP73, ESR2, TAB1, MAP 3K5, FN1, UBD, RUNX1, PIK3R2 and TNF, which might play an essential role in obesity associated type 2 diabetes mellitus was further screened. CONCLUSIONS: The present study could deepen the understanding of the molecular mechanism of obesity associated type 2 diabetes mellitus, which could be useful in developing therapeutic targets for obesity associated type 2 diabetes mellitus.

Key Vitamin D Target Genes with Functions in the Immune System.

The biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), modulates innate and adaptive immunity via genes regulated by the transcription factor vitamin D receptor (VDR). In order to identify the key vitamin D target genes involved in these processes, transcriptome-wide datasets were compared, which were obtained from a human monocytic cell line (THP-1) and peripheral blood mononuclear cells (PBMCs) treated in vitro by 1,25(OH)2D3, filtered using different approaches, as well as from PBMCs of individuals supplemented with a vitamin D3 bolus. The led to the genes ACVRL1, CAMP, CD14, CD93, CEBPB, FN1, MAPK13, NINJ1, LILRB4, LRRC25, SEMA6B, SRGN, THBD, THEMIS2 and TREM1. Public epigenome- and transcriptome-wide data from THP-1 cells were used to characterize these genes based on the level of their VDR-driven enhancers as well as the level of the dynamics of their mRNA production. Both types of datasets allowed the categorization of the vitamin D target genes into three groups according to their role in (i) acute response to infection, (ii) infection in general and (iii) autoimmunity. In conclusion, 15 genes were identified as major mediators of the action of vitamin D in innate and adaptive immunity and their individual functions are explained based on different gene regulatory scenarios.

Identification of CBF Transcription Factors in Tea Plants and a Survey of Potential CBF Target Genes under Low Temperature.

C-repeat binding factors (CBFs) are key signaling genes that can be rapidly induced by cold and bind to the C-repeat/dehydration-responsive motif (CRT/DRE) in the promoter region of the downstream cold-responsive (COR) genes, which play a vital role in the plant response to low temperature. However, the CBF family in tea plants has not yet been elucidated, and the possible target genes regulated by this family under low temperature are still unclear. In this study, we identified five CsCBF family genes in the tea plant genome and analyzed their phylogenetic tree, conserved domains and motifs, and cis-elements. These results indicate that CsCBF3 may be unique in the CsCBF family. This is further supported by our findings from the low-temperature treatment: all the CsCBF genes except CsCBF3 were significantly induced after treatment at 4 °C. The expression profiles of eight tea plant tissues showed that CsCBFs were mainly expressed in winter mature leaves, roots and fruits. Furthermore, 685 potential target genes were identified by transcriptome data and CRT/DRE element information. These target genes play a functional role under the low temperatures of winter through multiple pathways, including carbohydrate metabolism, lipid metabolism, cell wall modification, circadian rhythm, calcium signaling, transcriptional cascade, and hormone signaling pathways. Our findings will further the understanding of the stress regulatory network of CsCBFs in tea plants.

Vitamin D Signaling in the Context of Innate Immunity: Focus on Human Monocytes.

The vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) activates at sub-nanomolar concentrations the transcription factor vitamin D receptor (VDR). VDR is primarily involved in the control of cellular metabolism but in addition modulates processes important for immunity, such as anti-microbial defense and the induction of T cell tolerance. Monocytes and their differentiated phenotypes, macrophages and dendritic cells, are key cell types of the innate immune system, in which vitamin D signaling was most comprehensively investigated via the use of next generation sequencing technologies. These investigations provided genome-wide maps illustrating significant effects of 1,25(OH)2D3 on the binding of VDR, the pioneer transcription factors purine-rich box 1 (PU.1) and CCAAT/enhancer binding protein α (CEBPA) and the chromatin modifier CCCTC-binding factor (CTCF) as well as on chromatin accessibility and histone markers of promoter and enhancer regions, H3K4me3 and H3K27ac. Thus, the epigenome of human monocytes is at multiple levels sensitive to vitamin D. These data served as the basis for the chromatin model of vitamin D signaling, which mechanistically explains the activation of a few hundred primary vitamin D target genes. Comparable epigenome- and transcriptome-wide effects of vitamin D were also described in peripheral blood mononuclear cells isolated from individuals before and after supplementation with a vitamin D3 bolus. This review will conclude with the hypothesis that vitamin D modulates the epigenome of immune cells during perturbations by antigens and other immunological challenges suggesting that an optimal vitamin D status may be essential for an effective epigenetic learning process, in particular of the innate immune system.

Identification, expression, and putative target gene analysis of nuclear factor-Y (NF-Y) transcription factors in tea plant (Camellia sinensis).

MAIN CONCLUSION: Genome-wide identification and characterization of nuclear factor-Y family in tea plants, and their expression profiles and putative targets provide the basis for further elucidation of their biological functions. The nuclear factor-Y (NF-Y) transcription factors (TFs) are crucial regulators of plant growth and physiology. However, the NF-Y TFs in tea plant (Camellia sinensis) have not yet been elucidated, and its biological functions, especially the putative target genes within the genome range, are still unclear. In this study, we identified 35 CsNF-Y encoding genes in the tea plant genome, including 10 CsNF-YAs, 15 CsNF-YBs and 10 CsNF-YCs. Their conserved domains and motifs, phylogeny, duplication event, gene structure, and promoter were subsequently analyzed. Tissue expression analysis revealed that CsNF-Ys exhibited three distinct expression patterns in eight tea tree tissues, among which CsNF-YAs were moderately expressed. Drought and abscisic acid (ABA) treatment indicated that CsNF-YAs may have a greater impact than other subunit members. Furthermore, through the genome-wide investigation of the presence of the CCAAT box, we found that CsNF-Ys may participate in the development of tea plants by regulating target genes of multiple physiological pathways, including photosynthesis, chlorophyll metabolism, fatty acid biosynthesis, and amino acid metabolism pathways. Our findings will contribute to the functional analysis of NF-Y genes in woody plants and the cultivation of high-quality tea plant cultivars.

In vivo transcriptome changes of human white blood cells in response to vitamin D.

In the vitamin D intervention study VitDbol (NCT02063334) blood samples were drawn directly before an oral bolus (2000 µg vitamin D3) and 24 h later. The focus of phase II of VitDbol was the transcriptome-wide analysis of the effects of vitamin D gene expression in human peripheral blood mononuclear cells (PBMCs). All five participants responded in an individual fashion to the bolus by increases in serum levels of the vitamin D metabolites 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). RNA sequencing identified 15.040 commonly expressed genes in PBMCs, 702 (4,7%) of which were significantly (p < 0,05) affected by the vitamin D3 bolus. KEGG pathway analysis suggested that these genes are involved in general protein translation, monocyte differentiation and cellular growth control. Previously published transcriptome-wide studies in comparable cell systems confirmed 234 of the 702 vitamin D target genes, leaving many genes, such as HLA-A and HLA-C, as novel discoveries. Interestingly, in vivo stimulated PBMCs of this study showed a larger number of common vitamin D target genes with the monocytic cell line THP-1 than with in vitro stimulated PBMCs. The expression pattern of vitamin D target genes differed significantly between individuals and the average expression change can serve as a marker for vitamin D responsiveness. In conclusion, this study demonstrates that under in vivo conditions changes in 25(OH)D3 and 1,25(OH)2D3 serum concentrations alter the expression of more than 700 vitamin D target genes in human leukocytes.

Simultaneous miRNA and mRNA Transcriptome Profiling of Differentiating Equine Satellite Cells Treated with Gamma-Oryzanol and Exposed to Hydrogen Peroxide.

Gamma-oryzanol (GO) is a popular supplement for performance horses, dogs, and humans. Previous studies indicated that GO supplementation decreases creatine kinase activity and lactate level after exercise and may affect oxidative stress in Thoroughbred horses. GO may change genes expression in equine satellite cells (ESC). The purpose of this study was to evaluate the effect of GO on miRNA, gene expression, oxidative stress, and cell damage and viability in differentiating ESC pretreated with hydrogen peroxide (H2O2). ESCs were obtained from a young horse's skeletal muscle. ESCs were pre-incubated with GO (24 h) and then exposed to H2O2 for one hour. For the microRNA and gene expression assessment, the microarray technique was used. Identified miRNAs and genes were validated using real time-quantitative polymerase chain reaction. Several tests related to cell viability, cell damage, and oxidative stress were performed. The microarray analysis revealed differences in 17 miRNAs and 202 genes between GO-treated and control ESC. The tests related to apoptosis, cell viability, and oxidative stress showed that GO affects these processes to varying degrees. Our results suggest that GO can change miRNA and gene expression and may impact the processes involved in tissue repairing after an injury.

miRNA Expression Profile of Saliva in Subjects of Yang Deficiency Constitution and Yin Deficiency Constitution.

BACKGROUND/AIMS: Based on the theory of constitution in Traditional Chinese Medicine (TCM), the Chinese Han population has been classified into nine constitutions. Of these, Yang deficiency constitution mainly exhibit cold intolerance while Yin deficiency constitution mainly exhibit heat intolerance. Some studies have been carried out to explore the modern genetic and biological basis of such constitution classification, but more remains to be done. MicroRNA (miRNA) serves as post-transcriptional regulators of gene expression and may play a role in the classification process. Here, we examined miRNA expression profile of saliva to further improve the comprehensiveness of constitution classification. METHODS: Saliva was collected from Chinese Han individuals with Yang deficiency, Yin deficiency and Balanced constitutions (n=5 each), and miRNA expression profile was determined using the Human miRNA OneArray®v7. Based on 1.5 Fold change, means log2|Ratio|≥0.585 and P-value< 0.05, differentially expressed miRNA was screened. Target genes were predicted using DIANA-TarBasev7.0 and analysis of KEGG pathway was carried out using DIANA-mirPathv.3. RESULTS: We found that 81 and 98 differentially expressed miRNAs were screened in Yang deficiency and Yin deficiency constitution, respectively. Among them, 16 miRNAs were identical and the others were unique. In addition, the target genes that are regulated by the unique miRNAs were significantly enriched in 27 and 20 signaling pathways in Yang deficiency and Yin deficiency constitution, respectively. Thyroid hormone signaling pathway is present in both constitutions. These unique miRNAs that regulated target genes of thyroid hormone signaling pathway may be associated with cold intolerance or heat intolerance. CONCLUSION: The results of our study show that Yang deficiency and Yin deficiency constitutions exhibit systematic differences in miRNA expression profile. Moreover, the distinct characteristics of TCM constitution may be explained, in part, by differentially expressed miRNAs.

Vitamin D Genomics: From In Vitro to In Vivo.

The vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the exclusive high-affinity ligand of the vitamin D receptor (VDR), a transcription factor with direct effects on gene expression. Transcriptome- and epigenome-wide data obtained in THP-1 human monocytes are the basis of the chromatin model of vitamin D signaling. The model describes, how VDR's spatio-temporal binding profile provides key insight into the pleiotropic action of vitamin D. The transcription of some 300 primary target genes is significantly modulated through the action of genomic VDR binding sites in concert with the pioneer transcription factor PU.1 and the chromatin organizer CTCF. In parallel, the short-term vitamin D intervention study VitDbol (NCT02063334) was designed, in order to extrapolate insight into vitamin D signaling from in vitro to in vivo. Before and 24 h after a vitamin D3 bolus chromatin and RNA were prepared from peripheral blood mononuclear cells for epigenome- and transcriptome-wide analysis. The study subjects showed a personalized response to vitamin D and could be distinguished into high, mid, and low responders. Comparable principles of vitamin D signaling were identified in vivo and in vitro concerning target gene responses as well as changes in chromatin accessibility. In conclusion, short-term vitamin D supplementation studies represent a new type of safe in vivo investigations demonstrating that vitamin D and its metabolites have direct effects on the human epigenome and modulate the response of the transcriptome in a personalized fashion.

Identification and analysis of microRNAs-mRNAs pairs associated with nutritional status in seasonal sheep.

Given the important role of nutritional status for reproductive performance, we aimed to explore the potential microRNA (miRNA)-mRNA pairs and their regulatory roles associated with nutritional status in seasonal reproducing sheep. Individual ewes were treated with and without 0.3 kg/day concentrates, and the body condition score, estrus rate, and related miRNAs and target genes were compared. A total of 261 differentially expressed miRNAs were identified, including 148 hypothalamus-expressed miRNAs and 113 ovary-expressed miRNAs, and 349 target genes were predicted to be associated with nutritional status and seasonal reproduction in sheep. Ultimately, the miR-200b-GNAQ pair was screened and validated as differentially expressed, and a dual luciferase reporter assay showed that miR-200b could bind to the 3'-untranslated region of GNAQ to mediate the hypothalamic-pituitary-ovarian axis. Thus, miR-200b and its target gene GNAQ likely represent an important negative feedback loop, providing a link between nutritional status and seasonal reproduction in sheep toward enhancing reproductive performance and productivity.

Bonafide Targets of Deregulated microRNAs in Non-Small Cell Lung Cancer as Tool to Identify Novel Therapeutic Targets: A Review.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is an aggressive neoplasm with a poor survival and novel therapies are urgently needed. The study of deregulated micro- RNAs (dereg-miRs) could constitute a strategy helping to detect specific genes playing a relevant role in the disease. Thus, the oncoproteins encoded by these genes could be exploited as novel therapeutic targets to be inhibited by small molecules, aptamers, or monoclonal antibodies. METHODS: The present review is focused on candidate genes having convincing biological evidences to be both bona fide targets for dereg-miRs and playing a role in NSCLC progression. These genes were evaluated according to the molecular pathway they belong. Moreover, in the attempt to provide an even broader list of candidate therapeutic targets for NSCLC, the full list of genes was analyzed using the online tool Interactome DB. RESULTS: Among the identified targets, some of them belong to p53 or MAP kinase signaling pathways, and others include caspases, MCL1, and BCL2L2 (playing a role in apoptosis), ZEB1, ZEB2, and USP25 (epithelial-to-mesenchymal transition), EZH2, SOX9, and HOXA5 (differentiation), Paxillin, LIMK1 and MTDH (cytoskeleton remodeling), and HDGF (angiogenesis). In addition, other targets, such as TIMP-2, PIM-1, and components of the IGF-signaling pathways were suggested following the interactome analysis. CONCLUSION: Studies on dereg-miRs helped to identify a set of genes whose encoded proteins could constitute candidates for future therapeutic approaches.

Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) - a class-I KNOX gene in potato.

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato.