- Research article
- Open Access
Identification and characterization of novel and conserved microRNAs in several tissues of the Chinese rare minnow (Gobiocypris rarus) based on illumina deep sequencing technology
© Hong et al. 2016
- Received: 12 November 2015
- Accepted: 28 March 2016
- Published: 12 April 2016
MicroRNAs (miRNAs), which comprise a large family of endogenous small non-coding RNA molecules, play important roles in the regulation of gene expression in various biological processes. The Chinese rare minnow (Gobiocypris rarus) is a Chinese native fish species and is used extensively as an experimental fish in China; however, relevant biological data, especially miRNA transcriptome data, have not been well documented. To discover conserved and potential novel miRNAs in Chinese rare minnows, a pool of equal amounts of RNA obtained from 6 different adult rare minnow tissues (brain, eye, gill, liver, muscle and heart) was sequenced using illumina deep sequencing technology.
In the present study, 26,930,553 raw reads, representing 2,118,439 unique high-quality reads, were obtained from the pooled small RNA library. Using bioinformatics analysis, 352 conserved and 112 novel Chinese rare minnow miRNAs were first discovered and characterized in this study. Moreover, we found extensive sequence variations (isomiRs) in rare minnow miRNAs, including internal miRNA isomiRs and terminal isomiRs at both the 5′ and 3′ ends and nucleotide variants. Six conserved and 4 novel miRNAs were selected and validated in 6 different adult rare minnow tissues using quantitative real-time PCR (qPCR). The results showed that miR-30a, miR-30b, and Novel-37 are ubiquitously expressed in a variety of tissues. miR-16a, miR-9, miR-125b, miR-34a, and Novel-69 were predominantly expressed in the brain. Novel-115 and Novel-7 were highly expressed in gills, but were relatively weakly expressed in other tissues. These results provided the expression patterns of miRNA genes in Chinese rare minnow. Finally, based on bioinformatics predictions, we mainly found that Novel-94 and Novel-1b-5p were simultaneously targeted to the 3′UTR of Dmrt1, which controls sex determination and/or sexual differentiation in a variety of metazoans at different sites. Novel-29b targeted the 3′UTR of Foxl2, which is involved in the maintenance of ovarian function and the transcriptional regulation of gonadal differentiation-related genes. Novel-62 and Novel-53 targeted the 3′UTR of ERbeta1 and ERbeta2 (which regulate the transcription of target genes), respectively.
Rare minnow is a widely used model for assessing the risk of environmental pollution in China. Identifying and characterizing rare minnow miRNA genes is necessary to discover the biological function of miRNAs and to screen for new molecule biomarkers to assess the risk of environmental pollution in the future.
- Gobiocypris rarus
- Deep sequencing
- Target prediction
MicroRNAs (miRNAs), a class of endogenous, approximately 22-nt-long, small regulatory RNAs , play important gene-regulatory roles in various biological processes [2, 3] including cell differentiation [4–6], proliferation , growth [7, 8], and aging and apoptosis [9, 10]. Since the first miRNA lin-4 was characterized in C. elegans development , thousands of miRNAs have been found in animals and plants using genetic methods and through the sequencing of small RNA libraries [12, 13].
These small molecules can reduce the production of protein by modulating the stability and/or translational potential of their mRNA targets at the post-transcriptional level . Generally, miRNAs are thought to function mainly by binding to target mRNAs through imperfect base paring with the 3′ untranslated regions (UTRs) and recruit the RNA-induced silencing complex (RISC), finally leading to down-regulation of their target genes [1, 14, 15]. Currently, a large number of miRNAs have been found in a variety of organisms from worms to humans, suggesting the evolutionary conservation of miRNA regulation mechanisms [1, 9, 11]. For instance, the let-7 miRNA gene, which was initially identified as a significant regulator that is involved in the heterochronic pathway controlling developmental timing in C. elegans, was the first miRNA known to be well-conserved from nematodes to primates [16, 17]. It is estimated that more than 60 % of mRNAs exhibit conserved miRNA-binding sites in mammals . However, teleost miRNAs were first reported in zebrafish , and various miRNAs were found to play a role during zebrafish development; some of these functions have been characterized [19, 20]. Giraldez et al.  reported that miR-430 facilitates the deadenylation and clearance of maternal mRNAs during early zebrafish embryogenesis , and numerous studies have demonstrated the potential role of miRNAs in the regulation of tumorigenesis . For example, miR-125b is an important negative regulator of p53 and p53-induced apoptosis during development and the stress response in zebrafish and humans . Furthermore, through the combined use of miRNA microarray platforms and bioinformatics analysis, Craig et al.  identified changes in miRNA expression and related biological pathways in zebrafish following 7 days of exposure to fluoxetine (FLX). Furthermore, another study evaluated miRNAs in relation to toxin/chemical exposure in fish. Brzuzan et al. used qPCR to identify changes in the expression of 9 selected miRNAs in liver samples of whitefish following exposure to microcystin-LR (MC-LR) at a dose of 100 μg·kg−1 body weight for 24 or 48 h. Among these miRNAs, miR-122 exhibited the highest constitutive level, and this was associated with a decrease in the expression of genes coding for ferritin and a Ras-like protein .
The rare minnow (Gobiocypris rarus), a small freshwater cyprinid fish, is an endemic model organism that is mostly distributed in the upstream region of the Yangtze River, Sichuan Province, China. The fish are small (30–80 mm in length), are easy to culture in the laboratory, and have a relatively short life cycle, spawning hundreds of eggs with high fertilization and hatching rates [27, 28]. To date, this Cypriniformes species has been used extensively for various biological studies in China [29, 30]. Additionally, rare minnows have been widely applied in aquatic toxicity testing as a model vertebrate species for many years in our laboratory [27, 28, 30–34]. In the field of toxicology studies, several fish models are currently in use; however, research aimed at investigating miRNA alterations is mainly conducted in vitro, and in vivo studies remain in infancy . The discovery of miRNA genes in the rare minnow genome will contribute to a better understanding of the roles played by miRNAs in regulating diverse biological processes in fish. Such studies are expected to prove useful for the future screening of novel molecule biomarkers to assess the risk of environmental pollution and in vivo toxicity.
All fish were anesthetized with 2-phenoxyethanol before being euthanized. The fish were cared for in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals for the Science and Technology Bureau of China throughout the study.
The rare minnow has been maintained in our laboratory for over 13 years. The brood stock was maintained in a flow-through system based on dechlorinated tap water and was subjected to a 16:8 h light:dark cycle at 25 ± 1 °C . The brood stock was fed with newly hatched brine shrimp (Artemia nauplii) twice daily and granulated food (TetraMin, Tetra Werke, Melle, Germany) once daily. In this study, ten healthy adult rare minnows (5 months old) were selected for study. The average body weight and body length were 1.2 ± 0.4 g and 42.3 ± 3.7 mm, respectively.
Tissue samples including the brain, eye, gill, liver, muscle and heart were collected from adult rare minnows. All samples were immersed in liquid nitrogen immediately after collection and stored at −80 °C. Total RNA was isolated from the tissue samples using a mirVana™ microRNA Isolation Kit (Ambion, USA), according to the manufacturer’s instructions. RNA integrity was examined using the Agilent 2100 Bioanalyzer system (Santa Clara, CA, USA); the samples were then stored at −80 °C until analysis.
Small RNA library construction and sequencing
In this study, one miRNA library was constructed, and an equal quantity (10 μg) of total RNA was extracted from each of the various tissue samples. Briefly, 15–35 nt molecules were purified from total RNA (mirVana™ microRNA Isolation Kit, Ambion, USA) using polyacrylamide gel electrophoresis (PAGE); then, 5′ and 3′ adaptors (TruSeq® Small RNA Sample Preparation Kit, Illumina, USA) were ligated to the 5′ and 3′ termini of the RNAs, and the samples were used as templates for cDNA synthesis. Subsequently, PCR amplification was performed using primers that were complementary to both adaptors. After purification of the amplified cDNA constructs, the products were sequenced using HiSeq2500 technology. All sequencing reads were deposited in the National Center for Biotechnology Short Read Archive (SRA) database (http://www.ncbi.nlm.nih.gov/sra/) under the study accession SRP057175.
The original reads obtained from small RNA sequencing were processed by summarizing data production, evaluating sequencing quality and calculating the length distribution of the small RNA reads. The quality assessment and processing of sequenced reads were performed as recommended [36–38]. Briefly, the quality of the sequenced reads was assessed using the FastQC application (FastQC-0.11.2, http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/). Adapter sequences from the reads were removed with FASTX-toolkit (FASTX-toolkit 0.0.14, http://hannonlab.cshl.edu/fastx_toolkit/commandline.html). After adaptor trimming, filtering low quality tags and removing reads either < 18 nt or > 30 nt, the remaining reads were mapped to the zebrafish genome with a tolerance of one mismatch in the seed sequence. Subsequently, using BLAST against the Rfam (http://Rfam.sanger.ac.uk/) database, the reads that were mapped to the zebrafish genome were analyzed to annotate rRNA, tRNA, snRNA, snoRNA and other ncRNA sequences in the small sequences. Conserved rare minnow miRNAs in the sequences were then identified using BLAST searching against the miRNA database, miRBase (release 21, http://www.mirbase.org/) and no more than two mismatches can be allowed to identify homologs of known miRNAs. The sequences that did not match known miRNAs were mapped to the Ensembl (http://www.ensembl.org) zebrafish genome (Zv9) and rare minnow EST and GSS databases to identify potentially novel miRNA candidates; these were identified by folding the flanking genome sequence of unique small RNAs using MIREAP (https://sourceforge.net/projects/mireap/).
Validation of miRNAs by qPCR analysis
qPCR was used to validate six conserved and four novel selected miRNAs, and the relative expression levels were analyzed in six tissues from adult rare minnows, including the brain, liver, heart, muscle, eye and gill. Total RNA was isolated using the miRcute miRNA Isolation Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. Then, two micrograms of total RNA from each sample were reverse-transcribed into cDNA using the miRcute miRNA First-Strand cDNA Synthesis Kit (TIANGEN, Beijing, China). After this was completed, qPCR was performed using the miRcute miRNA qPCR Detection Kit (TIANGEN, Beijing, China). The following reaction solution was prepared on ice: 10 μL 2 × miRcute miRNA Premix (containing SYBR & ROX), 0.4 μL PCR forward primer (10 μM), 0.4 μL Uni-miR qPCR Primer (10 μM, part of the Kit), 1.6 μL cDNA, and RNase-free dH2O up to 20 μL. The reaction mixtures were incubated in a 96-well plate at 94 °C for 2 min followed by 40 cycles of 94 °C for 20 s and 60 °C for 34 s. All primers used in the reverse transcription and qPCR experiments are shown in Additional file 1: Table S1. Samples (n = 3) were run simultaneously for each gene in triplicate, and a non-template control was included in each plate. The relative miRNA expression level was calculated using the 2−ΔΔCt method after the threshold cycle (Ct); the 5S rRNA (submitted to the European Nucleotide Archive, http://www.ebi.ac.uk/ena/data/view/LN878980) was used as an endogenous control. miRNA expression levels are presented as 2−ΔΔCt mean ± SE (standard error), and error bars indicate the standard error of 2−ΔΔCt mean values.
Identification of Novel miRNA targets via computational analysis
Target prediction was performed by integrating four miRNA target prediction software packages, including miRanda (http://www.microrna.org/microrna/home.do) , PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_exe.html) , RNA22 (https://cm.jefferson.edu/rna22/)  and TargetScan (http://www.targetscan.org/fish_62/) . To obtain a better miRNA target analysis result, we used the local version of these four software packages. All newly identified miRNA sequences were used to query the ten gene sequences of rare minnows using the default parameters.
Illumina sequencing of small RNAs
Read statistics of the obtained small RNAs
High-quality reads (> =18 nt)
Aligned to the zebrafish genome
Conserved miRNAs in Metazoa
Non-conserved in Metazoa
Reads for novel miRNA prediction
In summary, the total number of sequence reads generated by high-throughput sequencing using the Illumina platform was of the same order of magnitude (107) as previous results for zebrafish ; however, this number was significantly different (106) from that obtained for Nile tilapia . These differences are due to the evolutionary relationships between the species; zebrafish and rare minnows are both cyprinids, whereas Nile tilapia belongs to cichlidae.
Conserved and Novel miRNAs in rare minnow
To identify conserved miRNAs in rare minnows, perfectly matched reads from the small RNA library were searched against Metazoa mature miRNAs in miRBase (Release 21). Alignment of the sequences to the miRBase database revealed that 732,306 of all sequences yielded a positive match to known miRNAs. Finally, we identified 352 conserved miRNAs (Additional file 2: Table S2) in rare minnow.
The 10 most frequent highly expressed novel miRNA candidates
Most expressed sequence
Abundant miRNAs play essential and broad regulatory function in biological processes. In the current study, the most highly expressed miRNA in rare minnows was miR-215 (51,777 reads). Previous studies have indicated that miR-215 is involved in a variety of cancers in mammals. For example, Deng et al. found that miR-215 is significantly up-regulated in gastric cancer tissues from either gastrectomy or gastroscopy by targeting retinoblastoma tumor-suppressor gene 1 (RB1) through its 3′-UTR in gastric cancer cells . Moreover, White et al. demonstrated that miR-215 overexpression decreased cellular migration and invasion in a renal cell carcinoma (RCC) cell line model using miRNA microarray and qPCR analyses .
Multiple isomiRs in the rare minnow
Validation and analysis of miRNA expression
Identification of Novel miRNA targets via computational analysis
This work represents an initial study on the miRNA profile of the Chinese rare minnow. The differential expression of miRNAs and the prediction of their target genes provide a basis for further understanding rare minnow miRNAs and the biological processes in which they are involved. The rare minnow is a model species for various biological studies in lower vertebrates in China. The identification and functional characterization of rare minnow miRNAs reported here will provide new opportunities for functional genome research on the rare minnow and should prove useful when screening for novel molecule biomarkers for use in assessing the risk of environmental pollution.
This work was supported by the Key Program of the National Natural Science Foundation of China (21437006), the Major International Joint Research Project of the National Natural Science Foundation of China (51420105012), and the Water Pollution Control and Treatment of the National Science and Technology Major Project (2014zx-07204-008-003).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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