Identification and characterization of known and novel microRNAs in three tissues of Chinese giant salamander base on deep sequencing approach.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although animal miRNAs have been extensively studied in model systems, less is known in other animal with limited genome sequence data, including Chinese giant salamander (Andrias davidianus). The identification of the full repertoire of miRNAs expressed in the liver, spleen and muscle of A. davidianus would significantly increase our understanding for physiological function of A. davidianus, in this ancient and endangered urodele amphibian. In this study, three independent small RNA libraries were constructed from the liver, spleen and muscle of A. davidianus. The libraries were subjected to high-throughput sequencing by using the Illumina deep sequencing. As a result, a total of 12,831,239, 13,592,195 and 9,887,531 raw reads representing 2,240,771, 1,363,266 and 1,964,252 clean reads per library were obtained separately. Through bioinformatics analysis, we identified total of 553 known miRNAs and 44 putative novel miRNAs in our small RNA dataset from liver, spleen and muscle tissues. Five known miRNAs (gga-miR-10a-5p, pma-miR-29d-5p, aca-miR-338-3p, hsa-miR-455-3p and ssa-miR-2184-5p_R-1) and three novel miRNAs (PC-5p-891_1763, PC-5p-32538_50 and PC-3p-33645_48) showed different expression in eight different tissues as revealed by stem-loop qPCR analysis. This study characterized the miRNA of A. davidianus for the first time, which provides an opportunity for further understanding of miRNA regulation function in A. davidianus ranked as living fossils.

Identification and characterization of microRNAs and their target genes from Nile tilapia (Oreochromis niloticus).

MicroRNAs (miRNAs) are a class of small single-stranded, endogenous 21-22 nt non-coding RNAs that regulate their target mRNA levels by causing either inactivation or degradation of the mRNAs. In recent years, miRNA genes have been identified from mammals, insects, worms, plants, and viruses. In this research, bioinformatics approaches were used to predict potential miRNAs and their targets in Nile tilapia from the expressed sequence tag (EST) and genomic survey sequence (GSS) database, respectively, based on the conservation of miRNAs in many animal species. A total of 19 potential miRNAs were detected following a range of strict filtering criteria. To test the validity of the bioinformatics method, seven predicted Nile tilapia miRNA genes were selected for further biological validation, and their mature miRNA transcripts were successfully detected by stem-loop RT-PCR experiments. Using these potential miRNAs, we found 56 potential targets in this species. Most of the target mRNAs appear to be involved in development, metabolism, signal transduction, transcription regulation and stress responses. Overall, our findings will provide an important foundation for further research on miRNAs function in the Nile tilapia.

IDENTIFCATION AND VALIDATION OF NOVEL microRNA MOLECULE FROM THE PELODISCUS SINENSIS BY BIOINFORMATICS APPROACHES.

MicroRNAs (miRNAs) are a group of endogenous, short non-coding RNAs with the length of about 22 nt, which mediate gene expression at the post-transcriptional level through mRNA degradation or translational repression. Among them, some are highly evolutionally conserved in the animal kingdom; this provides a powerful strategy for identifying miRNAs in new species. The Chinese soft-shelled turtle (Pelodiscus sinensis) is one of the most important freshwater aquaculture reptilian species in China, but related miRNAs have not been identified up to now. In the present study, a total of 10 Pelodiscus sinensis miRNAs were identified according to Pelodiscus sinensis ESTs and GSSs information in NCBI database by bioinformatics approaches. The RT-PCR-based assays were performed and found that 10 Pelodiscus sinensis miRNAs were expressed. Using these miRNAs, 22 target genes were identified. These genes encode 22 proteins involved in metabolism, signal transduction, transcriptional regulation, and development. These miRNAs and their targets will serve as useful resources for their, functional analyses in miRNA-regulated processes in Pelodiscus sinensis breeding and genetic research.

Maternal overweight and obesity and the risk of neural tube defects: a case-control study in China.

BACKGROUND: Studies have suggested that being obese before pregnancy is a risk factor for neural tube defects (NTDs), while results of many studies are controversial. This study aims to explore the association between maternal overweight and obesity before pregnancy and risk of NTDs. METHODS: A 1:1 matched case-control study was conducted. Cases were 459 women who delivered infants or conceived fetuses with NTDs in two provinces of China and controls were 459 women with live-born infants, without an apparent congenital malformation, matched with cases by region, delivery hospital, and year of childbirth. RESULTS: Compared with normal body weight, maternal obesity (body mass index, BMI ≥ 28 kg/m(2)) before pregnancy had a significant increased risk for NTDs as a group after adjusting for age, occupation, educational level, family income, parity, and use of folic acid (odds ratio, OR = 2.45; p < 0.05). No significant increase in NTD risk was found for maternal underweight (BMI < 18.5 kg/m(2)) and overweight (BMI from 24 to 28 kg/m(2)). For three subtypes of NTDs, anencephaly, spina bifida, and encephalocele, no significant increase in risk was found with maternal underweight, overweight, or obesity (all, p > 0.05). CONCLUSION: Maternal obesity before pregnancy is associated with risk of NTDs and should be considered in maternal reproductive health care.

Maternal body mass index and the association between folic acid supplements and neural tube defects.

AIM: To study the influence of maternal body mass index (BMI) on the association between folic acid supplementation and neural tube defects (NTDs) risk in offspring. METHODS: A hospital-based, case-control study was conducted between 2006 and 2008 on 459 mothers with NTDs-affected births and 459 mothers without NTDs-affected births. Logistic regression models examined the associations between folic acid supplementation and the NTDs risk in offspring for all mothers, underweight/normal weight mothers (BMI<24.0) and overweight/obese mothers (BMI ≥24.0). The effects were evaluated by adjusted odds ratio (AOR) and 95% confidence intervals (CIs) with SAS 9.1.3 software. RESULTS: The overall AOR for periconceptional folic acid supplementation was 0.315 (95% CI = 0.172-0.577) when compared with no supplements. Stratified by maternal BMI, the AOR for periconceptional folic acid supplementation in overweight/obese mothers was greater than in underweight/normal weight mothers (0.646 vs. 0.208). The AOR for folic acid supplementation within 3 months before conception was 0.711 (95% CI = 0.323-1.563) in all mothers. Stratified by maternal BMI, the AOR for folic acid supplementation within 3 months before conception in overweight/obese mothers was greater than in underweight/normal weight mothers (0.658 vs. 0.527). CONCLUSION: The association between folic acid supplementation and the reduced NTDs risk was weaker in overweight/obese mothers (BMI ≥24.0) than in underweight/normal weight mothers (BMI <24.0).

Previous abortion and the risk of neural tube defects: a case-control study.

OBJECTIVE: To further explore the association between abortion history and neural tube defect (NTD) risk and the association of different interpregnancy intervals from prior abortion to current conception on NTD risk. STUDY DESIGN: A matched case-control study was conducted. Cases were 459 women who delivered and gestated babies/fetuses with NTDs in two provinces of China and 459 women with live-born infants, without an apparent congenital malformation, matched with cases by the same region, the same hospital, and childbirth in the same year as controls. All of the subjects were investigated by trained interviewers with the same criteria. Conditional logistic regression models were used to calculate odds ratios (ORs) and their 95% CI of abortion history and interpregnancy intervals on NTD risk. RESULTS: Women with abortion history did not have an increased risk for spina bifida and encephalocele. The interpregnancy interval of < 6 months from prior abortion to current conception was significantly associated with anencephaly risk. CONCLUSION: Abortion with an interpregnancy interval of < 6 months from prior abortion to current conception may have an increased risk for anencephaly and should be considered in maternal reproductive health care.

A case-control study of the effects of pregnancy planning on neural tube defects and its primary preventive measures.

BACKGROUND: Measures for prevention of neural tube defects (NTDs) have been recommended for many years in China, but the compliance with these measures is unsatisfactory. This study aims to compare the effect differences between planned pregnancy and unplanned pregnancy in the compliance with these measures and analyze the interactions between pregnancy planning and these measures for NTD prevention. METHODS: A 1:1 matched case-control study was conducted. We randomly selected 349 women who delivered or gestated babies/fetuses with NTDs in the last two years in two provinces and matched them with 349 women who delivered babies without obvious birth defects as controls. RESULTS: In the case group, 99 women reported that they had planned their pregnancies, accounting for 28.4%, and the proportion who received preconception examinations and took folic acid prior to conception was 13.8 and 8.6%, respectively. According to the multivariate analysis, health education (odds ratio [OR], 0.350), preconception examinations (OR, 0.497) and folic acid consumption prior to conception (OR, 0.257) all had preventative effects on NTDs (for all, p < 0.05). In both groups, the proportions of women who received preconception examinations and reported folic acid intake were much higher for those who reported planning their pregnancies compared to women with an unplanned pregnancy (for all, p < 0.01); and for NTD prevention, synergistic interactions existed between pregnancy planning and the other preventive measures. CONCLUSION: Folic acid consumption prior to conception, preconception examinations, and health education have preventive effects on NTDs. Pregnancy planning can significantly promote compliance with these preventive behaviors. In addition, there are synergistic interactions between pregnancy planning and these measures.

Transcriptome analysis of the Chinese giant salamander (Andrias davidianus) using RNA-sequencing.

The Chinese giant salamander (Andrias davidianus) is an economically important animal on academic value. However, the genomic information of this species has been less studied. In our study, the transcripts of A. davidianus were obtained by RNA-seq to conduct a transcriptomic analysis. In total 132,912 unigenes were generated with an average length of 690 bp and N50 of 1263 bp by de novo assembly using Trinity software. Using a sequence similarity search against the nine public databases (CDD, KOG, NR, NT, PFAM, Swiss-prot, TrEMBL, GO and KEGG databases), a total of 24,049, 18,406, 36,711, 15,858, 20,500, 27,515, 36,705, 28,879 and 10,958 unigenes were annotated in databases, respectively. Of these, 6323 unigenes were annotated in all database and 39,672 unigenes were annotated in at least one database. Blasted with KEGG pathway, 10,958 unigenes were annotated, and it was divided into 343 categories according to different pathways. In addition, we also identified 29,790 SSRs. This study provided a valuable resource for understanding transcriptomic information of A. davidianus and laid a foundation for further research on functional gene cloning, genomics, genetic diversity analysis and molecular marker exploitation in A. davidianus.

Characterization of conserved and novel miRNAs using deep sequencing and prediction of miRNA targets in Crucian carp (Carassius auratus).

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of -22 nucleotides that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. However, little is known about identification of miRNAs and their target genes in C. auratus. In the present study, a small RNA library from pooled tissue of C. auratus was constructed and sequenced using the deep sequencing. A total of 320 conserved miRNAs (belonging to 105 families) as well as 11 potentially novel miRNAs were identified. Stem-loop qRT-PCR analysis confirmed that both conserved and novel miRNAs were expressed in C. auratus, and some of them were preferentially expressed in certain tissues. Subsequently, a total of 1668 potential target genes were predicted for these identified miRNAs and GO analysis showed that most of the targets were involved in lots of physiological actions. This study represents a first large-scale identification and characterization of C. auratus miRNAs and their potential target genes. Taken together, our results add new information to existing data on C. auratus miRNAs and should be useful for investigating the biological functions of miRNAs in fishes and other aquatic species.

Identification and characterization of the Chinese giant salamander (Andrias davidianus) miRNAs by deep sequencing and predication of their targets.

MicroRNAs (miRNAs) are non-coding small RNA of approximately 22 nt in length. They are considered as key regulators for gene expression at the post-transcriptional level and play an important role in the regulation of many fundamental biological processes. Andrias davidianus, as one of the largest amphibian on earth, may represent the transitional type of animal from aquatic to terrestrial life, but so far, no miRNA has been identified in the species. In this study, Illumina deep sequencing was used for high-throughput analysis of miRNAs in a pooled small RNA library isolated from eight tissues sample of A. davidianus including the spleen, liver, muscle, kidney, skin, testis, gut, and heart. In total, 6,213,146 reads were obtained, 5,962,175 of which were related to 143 unique miRNAs, including 140 conserved and three novel A. davidianus-specific miRNAs. Among them, seven conserved miRNAs and one novel miRNA were selected to validate their expression pattern by stem-loop qRT-PCR. Moreover, 4700 potential target genes were predicted for 143 A. davidianus miRNAs; GO enrichment analysis and KEGG pathway showed that most of the targets are involved in diverse biological processes including ubiquitin-mediated proteolysis, FOXO signaling pathway, tight junction, and spliceosome. This study provides the first large-scale identification and characterization of A. davidianus miRNAs, and predicted their potential target genes; it will lay a valuable foundation for future understanding the role of these miRNAs on regulating diverse biological processes.

Sequencing and de novo transcriptome assembly of the Chinese giant salamander (Andrias davidianus).

Next-generation technologies for determination of genomics and transcriptomics composition have a wide range of applications. Andrias davidianus, has become an endangered amphibian species of salamander endemic in China. However, there is a lack of the molecular information. In this study, we obtained the RNA-Seq data from a pool of A. davidianus tissue including spleen, liver, muscle, kidney, skin, testis, gut and heart using Illumina HiSeq 2500 platform. A total of 15,398,997,600 bp were obtained, corresponding to 102,659,984 raw reads. A total of 102,659,984 reads were filtered after removing low-quality reads and trimming the adapter sequences. The Trinity program was used to de novo assemble 132,912 unigenes with an average length of 690 bp and N50 of 1263 bp. Unigenes were annotated through number of databases. These transcriptomic data of A. davidianus should open the door to molecular evolution studies based on the entire transcriptome or targeted genes of interest to sequence. The raw data in this study can be available in NCBI SRA database with accession number of SRP099564.

Prediction and characterization of microRNAs from eleven fish species by computational methods.

MicroRNAs (miRNAs) are a family of single-stranded RNA molecules about 22 nt in length, which can regulate protein-coding gene expression in various organisms by post-transcriptional repression of messenger. In this research, the potential miRNAs and their target genes were analyzed and predicted by computational methods from the EST and GSS databases of eleven fish species, 43 potential miRNAs were identified, they belong to 38 miRNA families, some miRNAs are highly conserved in animal kingdom, the predicted target genes are involved in development, signal transduction, response to environmental stress and pathogen invasion. Taken together, our data suggest that there are a plentiful of miRNAs in these eleven fish species, these miRNAs may play some important roles by regulating their target genes, and the data provide important information for further functional studies.

Nausea and vomiting in early pregnancy and the risk of neural tube defects: a case-control study.

There has been considerable professional debate on the association between nausea and vomiting in early pregnancy (NVP) and neural tube defects (NTDs) risk. This study explored the association between NVP and NTDs risk, and the effect of folic acid supplements on the association. A 1:1 matched case-control study was conducted and conditional logistic regression model was used to analyze the associations. The result showed the odds ratio (OR) of severe NVP for NTDs was 2.403 (95%CI 1.437,4.017; P<0.001) and that of moderate NVP was 1.469 (95%CI 1.063,2.031; P = 0.020) compared with light NVP when adjusted by the potential confounders. Stratified by intake of folic acid supplements, the ORs for severe and moderate NVP turned to 2.147 (95%CI 1.140, 4.043; P = 0.018) and 2.055 (95%CI 1.320, 3.199; P = 0.001) in the stratum of non-intake of folic acid supplements while ORs reduced to 1.851 (95%CI 0.729, 4.699; P = 0.195) and 1.003 (95%CI 0.594, 1.694; P = 0.992) in the stratum of intake of folic acid supplements, respectively. We conclude that severe/moderate NVP has an association with the risk of NTDs, which was not found in the group with intake of folic acid supplements. Folic acid supplements should be recommended to use for the prevention of NTDs.

Identification of microRNA genes and their mRNA targets in Festuca arundinacea.

MicroRNAs (miRNAs) have emerged as a novel class of endogenous, small, non-coding RNAs of 22 nucleotides (nts) in length, which plays important roles in post-transcriptional degradation of target mRNA or inhibition of protein synthesis through binding the specific sites of target mRNA. Growing evidences have shown that miRNAs play an important role in various biological processes, including growth and development, signal transduction, apoptosis, proliferation, stress responses, maintenance of genome stability, and so on. In our study, we used bioinformatic tools to predict miRNA and the corresponding target genes of Festuca arundinacea. We used known miRNAs of other plants from miRBase to search against expressed sequence tags (EST) databases and genome survey sequences (GSS) of F. arundinacea. A total of 8 potential miRNAs were predicted. Phylogenetic analysis of the predicted miRNAs revealed that miRNA398c of F. arundinacea species was evolutionary highly conserved with Populus trichocarpa. The 8 potential miRNAs corresponding to 20 target genes were found. Most of the miRNA target genes were predicted to encode transcription factors that regulate cell growth and development, signaling, metabolism, and other biology processes. By bioinformatics methods, we can effectively predict novel miRNAs and its target genes and add information to F. arundinacea miRNA database. Moreover, it shows a path for the prediction and analysis of miRNAs to those species whose genomes are not available through bioinformatics tools.

Computational identification of microRNAs and their targets in perennial Ryegrass (Lolium perenne).

MicroRNAs (miRNAs) are small non-coding RNA molecules of 22 nucleotides in length that have been characterized as regulators of messenger RNA (mRNA) regulating a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. miRNAs have been extensively studied in various plant species; however, few information are available about miRNAs in perennial ryegrass, animal feed, and industrial raw materials. In this study, the 12 potential perennial ryegrass miRNAs were identified for the first time by computational approach. Using the newly identified miRNA sequences, the perennial ryegrass mRNA database was further used for BLAST search and detected 33 potential targets of miRNAs. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. Our result should be useful for further investigation into the biological functions of miRNAs in perennial ryegrass. The selected miRNAs representing four families were verified by RT-PCR experiment, indicating that the prediction method that we used to identify the miRNAs was effective.