ISSN 0006-2979, Biochemistry (Moscow), 2018, Vol. 83, No. 6, pp. 766-777. © Pleiades Publishing, Ltd., 2018. Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM17-498, March 26, 2018. Identification and Characterization of MicroRNAs in Skin of Chinese Giant Salamander (Andrias davidianus) by the Deep Sequencing Approach Yong Huang1,a,b* and Wang Bao Gong2 1College of Animal Science and Technology, Henan University of Science and Technology, 471023 Luoyang, China 2Key Laboratory of Tropical and Subtropical Fishery Resource Application and Cultivation, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China ae-mail: [email protected] be-mail: [email protected] Received November 9, 2017 Revision received January 14, 2018 Abstract—MicroRNAs (miRNA) play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. However, the miRNAs from skin of Andrias davidianus have not been reported. In this study, a small-RNA cDNA library was constructed and sequenced from skin of A. davidianus. A total of 513 conserved miRNAs belonging to 174 families were identified. The remaining 108 miRNAs we identified were novel and likely to be skin tissue-specific but were expressed at low levels. The presence of randomly selected 15 miRNAs identified and their expression in eight different tissues from A. davidianus were validated by stem-loop qRT-PCR. For better understanding the functions of miRNAs, 129,791 predicated target genes were analyzed by GO and their pathways illustrated by KEGG path- way analyses. The results show that these identified miRNAs from A. davidianus skin are involved in a broad range of phys- iological functions including metabolism, growth, development, and immune responses. This study exhaustively identifies miRNAs and their target genes, which will ultimately pave the way for understanding their role in skin of A. davidianus and other amphibians. Further studies are necessary to better understand miRNA-mediated gene regulation. DOI: 10.1134/S0006297918060147 Keywords: Andrias davidianus, microRNA, skin, deep sequencing, target gene MicroRNAs (miRNAs) are small noncoding tran- miRNAs play important regulatory roles in numerous scripts of ~22 nucleotides (nt) in length that can regulate biological processes, including organ development, cell expression at the posttranscriptional level, which leads to differentiation, proliferation, apoptosis, homeostasis, the inhibition of translation or the degradation of mRNAs metabolism, tumorigenesis, bacterial and viral infections, [1, 2]. Mature miRNA genes are transcribed from pri- immunological regulation, etc. in plants, animals, and miRNAs that can form a fold-back, hairpin structure. In even viruses [6-10]. a sequential process, the pre-miRNAs are exported into The Chinese giant salamander (Andrias davidianus) the cytoplasm and cleaved by RNase III Dicer and form belonging to the family Cryptobranchidae Fitzinger 1826 a double-stranded RNA [3, 4]. One of the duplex strands is the largest amphibian in the world [11]. It has a fair- of the mature miRNAs can be incorporated into the sounding Chinese name, “babyfish (wawa-yu)”, and it RISCs; thereafter, they bind to the 3′ UTR of the target mainly inhabits mountain brooks or interior water areas mRNAs to regulate expression of target mRNAs [5]. An in South-Western and Eastern China. Andrias davidianus increasing number of studies have demonstrated that is renowned as a living fossil, since it has been in existence for more than 350 million years, representing the transi- Abbreviations: GO analysis, gene ontology enrichment analysis; tional type of animal from aquatic to terrestrial life [12, KEGG analysis, Kyoto Encyclopedia of Genes and Genomes 13]. So, A. davidianus is also considered to be a valuable pathway analysis; miRNA, microRNA. model of scientific value, medical value, edible value, * To whom correspondence should be addressed. view value, and so on [14]. However, due to habitat loss, 766 miRNAs FROM SKIN OF CHINESE GIANT SALAMANDER: DEEP SEQUENCING 767 climate change, environment pollution, overfishing, and Small RNA library construction and deep sequencing. disease (such as bacterial, fungal, and viral infections), Total RNA was isolated from the above-described sample their populations have dramatically declined, making it using an RNAiso Plus kit (Takara, China) according to an endangered species [15, 16]. It is now estimated to be the manufacturer’s instructions. The integrity of the total critically endangered by the International Union for RNA was analyzed using an Agilent 2100 Bioanalyzer Conservation of Nature and Nature Resources and is a system and RNA6000 Nano LabChip Kit (Agilent, USA) State Special Protected Animal (category II) under with RNA integrity value (RIN) >8.0. Then a small RNA Chinese Conservation Law, and in Appendix I of the library was constructed respectively using TruSeq Small Convention on International Trade in Endangered RNA Sample Prep Kits (Illumina, USA). Briefly, about Species of Wild Fauna and Flora. total 5 µg RNA was resolved on denaturing PAGE gels, In recent years, with the rapid development of artifi- and then fractions 18 to 26 nt in size were collected. Two cial cultivation, more than two million A. davidianus are adaptors were sequentially ligated to the 5′ and 3′ ends of bred annually, which become an important commercial purified small RNAs. The ligation products were reverse- aquaculture species in China. However, with the increase transcribed using SuperScript Reverse Transcriptase II of intensive culture of this species, emerging infectious (Life Technologies, USA) and amplified with 15 PCR diseases have been increasing and have caused major cycles. Then, 8% (w/v) PAGE was used to purify the impacts to A. davidianus industry. The skin of A. davidi- amplification products. Finally, the library was used for anus is a very important immune organ and is considered clustering and sequencing using an Illumina Genome to be the first line of defense against external pathogenic Analyzer II (Hangzhou, China). After image analysis, microbes or predators, belonging to the nonspecific sequence quality evaluation and summarization of data immune defense system [17, 18]. Therefore, understand- production were performed with the Illumina Pipeline. ing the mechanisms of the immune systems of A. davidi- The raw small RNA transcriptome sequencing data from anus in response to bacterial and viral infection is essen- the skin of A. davidianus was submitted to the NCBI tial for managing disease outbreaks and may contribute to Sequence Read Archive under accession number the sustained development of A. davidianus culture. So, SRP110836. identification, expression analysis, and target gene predi- Sequence analysis and miRNA identification. The raw cation of the miRNAs in the skin should facilitate our sequences were filtered to remove the adapter sequences understanding of the involvement of miRNA in immune and contaminated reads. The retained mappable reads system functions. There are reports concerning the were further analyzed following the work flow using miRNAs of A. davidianus [14, 19, 20]. In these previous ATCG101-miRsoftware (version 4.2; LC Sciences). The reports, researchers identified miRNAs from the muscle, unique small RNA sequences were firstly mapped to spleen, liver, testis, ovary, and mixed tissues of A. davidi- known pre-miRNAs deposited in the miRBase database anus, but no study on miRNAs from skin of A. davidianus (release 21.0) (http://www.mirbase.org/) using the map- has been reported. Therefore, in the present study, we ping program Bowtie (http://bowtie-bio.sourceforge.net/ constructed a small-RNA cDNA library from A. davidi- index.shtml). After that, the reads matched to rRNA, anus skin. Through deep sequencing of the small RNA tRNA, snRNA, snoRNA and other noncoding RNA library and subsequent bioinformatics analysis, miRNAs sequences deposited in Rfam (http://rfam.janelia.org) in skin of A. davidianus have belen identified. and the repeat-RepBase (http://www.girinst.org/repbase) were deducted. The remaining reads were mapped onto the reference transcriptomic dataset of A. davidianus [21]. MATERIALS AND METHODS Sequences without mismatch were retained for novel miRNA prediction. Finally, candidate novel miRNAs Animal samples collection. Healthy A. davidianus were identified by prediction of the secondary structures individuals were obtained from a farm located in Luan with the Mfold program (http://mfold.rna.albany.edu/ Chuan (Henan Province, China). Skin tissue was collect- ?q=mfold/RNA-Folding-Form). The criteria for sec- ed from a five-year-old A. davidianus salamander imme- ondary structure prediction were: (i) number of diately after dissection and washed in sterile PBS. A sam- nucleotides in one bulge in stem, 12; (ii) number of base ple for RNA extraction was snap-frozen in liquid nitrogen pairs in the stem region of the predicted hairpin, 16; (iii) and stored at −80°C before use. The present study involv- cutoff of free energy (kcal/mol), 15; (iv) length of hair- ing animals was conducted according to the Regulations pin (up and down stems + terminal loop), 50; (v) length for the Administration of Affairs Concerning Experi- of hairpin loop, 20; (vi) number of nucleotides in one mental Animals (Ministry of Science and Technology, bulge in mature region, 8; (vii) number of biased errors