A pregnancy diagnosis is an important standard for control of livestock’s reproduction in paricular dairy cattle. High reproductive performance in dairy animals is a essential condition to realize of high life-time production. Pregnancy diagnosis is crucial to shortening the calving interval by enabling the farmer to identify open animals so as to treat or re-breed them at the earliest opportunity. MicroRNAs are short RNA molecules which are critically involved in regulating gene expression during both health and disease. This study is sought to establish the feasible of circulating miRNAs as biomarkers of early pregnancy in cattle. We applied Illumina small-RNA sequencing to profile miRNAs in plasma samples collected from 12 non-pregnant cows (“open” cows: samples were collected before insemination (non-pregnant state) and after pregnancy check at the indicated time points) on weeks 0, 4, 8, 12 and 16. Using small RNA sequencing we identified a total of 115 miRNAs that were differentially expressed weeks 16 relative to non-pregnancy (“open” cows). Weeks 8, 12 and 16 of pregnancy commonly showed a distinct increase in circulating levels of miR-221 and miR-320a. Through genome-wide analyses we have successfully profiled plasma miRNA populations associated with pregnancy in cattle. Their application in the field of reproductive biology has opened up opportunities for research communities to look for pregnancy biomarker molecules in dairy cattle.

The carcinogenesis mechanism of human salivary gland adenocarcinoma NOS is poorly understood. MicroRNA155(miRNA155) has been involved in the carcinogenesis of many malignant tumors. The purpose of this study was to examine the role of miRNA155 in tumor growth and invasion of adenocarcinoma NOS. Using SGT cells as a model for adenocarcinoma NOS, cell proliferation was examined by MTT assay after knocking down miRNA155 expression, and cell cycle analysis was performed. Invasive capacity by a Transwell culture assay, and miRNA155 expression in SGT cell line by RT-PCR were examined. In MTT assay, proliferation of SGT-miRNA155 cells was decreased prominently after 96 hrs. Proliferation of SGT cells was markedly inhibited by knocking down miRNA155, resulting from a blockade of cell cycle in the G1 phase, but apoptosis was increased about 4 folds. In adhesion assay, SGT-miRNA155 cells decreased about 60% compared to SGT cells. In invasion assay, inhibition of miRNA155 significantly suppressed the invasive capacity of about 34% SGT cells. mRNA expression of SGT-miRNA155 cells prominently were decreased compared to SGT cells by RT-PCR. It suggested that miRNA155 could play an role in cell cycle progression and invasion in SGT cells, including antitumor effect. These results have provided insights into the carcinogenic mechanisms and new intervention method of salivary gland adenocarcinoma NOS.

MicroRNAs (miRNAs) are approximately 22 nucleotides of small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The miRNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, metabolism, imprinting, and differentiation. Recently, a paper has shown that expression of the miRNA-302/367 cluster expressed abundantly in mouse and human embryonic stem cells (ESCs) can directly reprogram mouse and human somatic cells to induced pluripotent stem cells (iPSCs) efficiently in the absence of any of the four factors, Oct4, Sox2, c-Myc, and Klf4. To apply this efficient method to porcine, we analyzed porcine genomic sequence containing predicted porcine miRNA-302/367 cluster through ENSEMBL database, generated a non-replicative episomal vector system including miRNA-302/367 cluster originated from porcine embryonic fibro-blasts (PEF), and tried to make porcine iPSCs by transfection of the miRNA-302/367 cluster. Colonies expressing EGFP and forming compact shape were found, but they were not established as iPSC lines. Our data in this study show that pig miRNA-302/367 cluster could not satisfy requirement of PEF reprogramming conditions for pluripotency. To make pig iPSC lines by miRNA, further studies on the role of miRNAs in pluripotency and new trials of transfection with conventional reprogramming factors are needed.

MicroRNAs (miRNAs, miRs) are about 21-25 nucleotides in length and regulate mRNA translation by base pairing to partially complementary sites, predominantly in the 3’-untranslated region (3’-UTR) of the target mRNA. In this study, the expression profile of miRNAs was compared and analyzed for the establishment of miRNA-related odontoblast differentiation using MDPC-23 cells derived from mouse dental papilla cells. To determine the expression profile of miRNAs during the differentiation of MDPC-23 cells, we employed miRNA microarray analysis, quantitative real-time PCR (qRT-PCR) and Alizaline red-S staining. In the miRNA microarray analysis, 11 miRNAs were found to be up- or down-regulated more than 3-fold between day 0 (control) and day 5 of MDPC-23 cell differentiation among the 1,769 miRNAs examined. In qRT-PCR analysis, the expression levels of two of these molecules, miR-194 and miR-126, were increased and decreased in the control MDPC-23 cells compared with the MDPC-23 cells at day 5 of differentiation, respectively. Importantly, the overexpression of miR-194 significantly accelerated mineralization compared with the control cultures during the differentiation of MDPC-23 cells. These results suggest that the miR-194 augments MDPC-23 cell differentiation, and potently accelerates the mineralization process. Moreover, these in vitro results show that different miRNAs are deregulated during the differentiation of MDPC-23 cells, suggesting the involvement of these genes in the differentiation and mineralization of odontoblasts.

MicroRNAs are ~22nt small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. Micro RNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, control of metabolic pathways, imprinting and differentiation. The expression of miRNAs is often regulated in tissue specific and developmental stage‐specific manners. More than 500 miRNAs have been reported in diverse eukaryotic organism so far. One of the biological functions of miRNAs seems to be the regulation of self‐renewal versus differentiation in stem cells. Recent efforts have focused on defining the miRNA expression profile in undifferentiated ESCs as compared to their differentiated progeny. Among the so‐called ES‐specific miRNAs, the 302‐367 cluster stands out due to its intracellular abundance and high cell type specificity. Levels of miRNA 302‐367 correlate with Oct4 transcripts in ESCs and early embryonic development, indicating an important role in ESC homeostasis and maintenance of pluripotency. Several months ago, a paper showed that expression of the miRNA 302‐367 cluster can directly reprogram mouse and human somatic cell to an iPS cell in absence of any of the four factors (Oct4, Sox2, c‐Myc, Klf4) efficiently. To apply this efficient method to porcine, we made an inducible vector system including miRNA 302‐367 cluster originated from porcine embryonic fibroblasts and could make porcine ips by the miRNA 302‐367 cluster.

Periodontitis results from the activation of host immune and inflammatory defense responses to subgingival plaque bacteria, most of which are gram-negative rods with lipopolysaccharides (LPSs) in their cell walls. LPSs have been known to induce proinflammatory responses and recently it was reported also that they induce the expression of microRNAs(miRNAs) in host cells. In our current study therefore, we aimed to examine and compare the miRNA expression patterns induced by the LPSs of major periodontopathogens in the human gingival epithelial cell line, Ca9-22. The cells were treated with 1 μg/ml of E. coli (Ec) LPS or 5 μg/ml of an LPS preparations from four periodontopathogens Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn) for 24 h. After small RNA extraction from the treated cells, miRNA microarray analysis was carried out and characteristic expression profiles were observed. Fn LPS most actively induced miRNAs related to inflammation, followed by Aa LPS, Pi LPS, and Ec LPS. In contrast, Pg LPS only weakly activated miRNAs related to inflammation. Among the miRNAs induced by each LPS, miR-875-3p, miR-449b, and miR-520d-3p were found to be commonly up-regulated by all five LPS preparations, although at different levels. When we further compared the miRNA expression patterns induced by each LPS, Ec LPS and Pi LPS were the most similar although Fn LPS and Aa LPS also induced a similar miRNA expression pattern. In contrast, the miRNA profile induced by Pg LPS was quite distinctive compared with the other bacteria. In conclusion, miR-875- 3p, miR-449b, and miR-520d-3p miRNAs are potential targets for the diagnosis and treatment of periodontal inflammation induced by subgingival plaque biofilms. Furthermore, the observations in our current study provide new insights into the inflammatory miRNA response to periodontitis.

om llnique miRNA encoding genes and also from intergene seqllences. 80 far it is generally suggestecl that one miRNA could target multiple genes. and also several miRNAs could be orchestrated to downregulate a s pecific gene. However. there would be many possibilities to procluce more miRNAs and to target gene in more compli catecl manners tha n we expected Here‘ we developed a detection program of miRNA consensus sequences from non- cocling region of genetic corcls As the ha irpin structure 01' tRNA has step-loop like hybridi zation by a single strand RNA. the repeated 3-5 nt symmetric arrangement with 6- 12 nt spanning sequences for hail‘pin head is necessary to form a preCllrsor rruRNA The DNA base pair‘ polarity program symbolizecl by the electrostatic polarities of pyrimidine and purine is now reinforcecl by the cletection program fOI symmetric DNA strand from non-coding regions. For example, we iclentifi ecl candidate miRNAs targeting eIF5A mRNA (miR-eIF5A-l)‘ 288 ribosomal RNA (miR- 28S-1). and dentin sialophosphate protein mRNA (miR- D8PP-1). 1n t his study the detection methocl of 288 ribosomal RNA was demonstrated ancl the molecular biological effects 01' miRNA targeting in vitro culture system were evaluatecl by miRNA RT• PCR, Northen blot, siRNA interference, and in situ hybridization. We firstly sea rched the candidate miRNA from the intron sequences of each objectiγe gene using the programs of syrrunetric sequence cletection a ncl DNA base pair polarity, ancl secondarily siRNA procluced by in vitro transcription and inclucible lenti virus vector cons tru ction was transfectecl into HEK293 cells. The transfectecl siRNA of miR-28S-1 was accumulated in the nucleoli ancl inclllced apoptosis extens ively in 2 days cultur8. Northern blot using miR-288-1 probe showed strong reaction in t he 288 bancJ of total RNA and also showed a smeared band in small size representing the presence of t he precursor and mature miRNAs 이 miR-288-1. In in situ hyridi zation most of cells revealed intensive miR-288-1 positive reaction in their cytopl asms. mirni cking t he abllndant localization of 288 RNA From the above study. we presume that rniRNAs targeting s pecific genes are possibly derivecl from the in tron seq uences of the objective gene, and suggest that the symmetirc sequence detect ion ancl DNA base pair polarity program is useflll to define the candiclate miRNA

The THO/TREX complex mediates the transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and it has a role in small RNA-dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, whichencodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only the levels of siRNAs, but also of mature miRNAs were reduced in tho2 mutants. As a consequence miRNA target mRNAs accumulated to higher levels than in wild type. Yeast two hybrid experiments showed that THO2 does not seem to interact with any of the known miRNA biogenesis components, implying a more indirect role of THOs in small RNA biogenesis. We also detected alterations in the splicing pattern of genes encoding Serine/Arginine-rich proteins in tho2 mutants, suggesting a previously unappreciated role of the THO/TREX complex in alternative splicing.

MicroRNAs (miRNAs) are a class of non-coding RNAs of approximately 21-nt which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, the miRNAs and their target genes remain largely unknown in pepper, one of the most important crops cultivated worldwide. Here we employed high-throughput small RNA sequencing to extensively identify miRNAs in pepper from 10 different libraries, including leaf, stem, root, flower, and six developmental stage fruits. Based on bioinformatics pipeline, we successfully identified 29 and 35 families of conserved and novel miRNAs, respectively. We noticed that some miRNAs, whose targets were validated experimentally in this study, exhibited prominent changes in expression levels during fruit development stages. From the qRT-PCR analysis of the target mRNAs, including the SBP-transcription factor and F-box protein, we found that expression of these two target mRNAs gradually decreased in general during fruit development and was negatively correlated with the expression of their corresponding miRNAs. The validation of miRNA-directed cleavage of these target mRNAs, combined with the results of qRT-PCR analysis, likely suggests that some miRNAs in pepper may play a role in fruit development. Conclusively, our study first utilizes high-throughput sequencing to identify and characterize conserved and novel miRNAs and their targets in pepper, providing a basis for understanding the functional roles of miRNAs in pepper.

MicroRNAs (miRNAs) function as a key regulator of diverse cellular functions. To find out novel miRNAs that promote the differentiation of mouse embryonic stem cells (mESCs), we compared the miRNAs expression profiles of mESCs under self-renewal vs. differentiation states. We noticed that miR-222 was highly expressed during the differentiation of mESCs. Quantitative RT-PCR analysis revealed that expression of miR-222 was up-regulated during the embryonic bodies formation and retinoic acid -dependent differentiation. When miR-222 was suppressed by antogomiR-222, the differentiation of mESCs was delayed compared to control. Self-renewal marker expression or cell proliferation was not affected but the expression of lineage specific marker was suppressed by the treatment of miR-222 inhibitor during the differentiation of mESCs. Taken together, these results suggest that miR-222 functions to promote the differentiation of mESCs by regulating expression of differentiation related genes.