MicroRNA and piRNA Profiles in Normal Human Testis Detected by Next Generation Sequencing

et al. (2013) MicroRNA and piRNA Profiles in Normal Human Testis Detected by Next Generation Sequencing. PLoS ONE 8(6): e66809. doi:10.1371/journal.pone.0066809 MicroRNA and piRNA Profiles in Normal Human Testis Detected by Next Generation Sequencing Qingling Yang 0 Juan Hua 0 Liu Wang 0 Bo Xu 0 Huan Zhang 0 Nan Ye 0 Zhiqiang Zhang 0 Dexin Yu 0 Howard J. Cooke 0 Yuanwei Zhang 0 Qinghua Shi 0 Wei Yan, University of Nevada School of Medicine, United States of America 0 1 Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China , Hefei, Anhui , China , 2 Department of Urology, the First/Second/Fourth Affiliated Hospital, Anhui Medical University , Hefei, Anhui , China , 3 MRC Human Genetics Unit and Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh , United Kingdom Background: MicroRNAs (miRNAs) are the class of small endogenous RNAs that play an important regulatory role in cells by negatively affecting gene expression at transcriptional and post-transcriptional levels. There have been extensive studies aiming to discover miRNAs and to analyze their functions in the cells from a variety of species. However, there are no published studies of miRNA profiles in human testis using next generation sequencing (NGS) technology. Results: We employed Solexa sequencing technology to profile miRNAs in normal human testis. Total 770 known and 5 novel human miRNAs, and 20121 piRNAs were detected, indicating that the human testis has a complex population of small RNAs. The expression of 15 known and 5 novel detected miRNAs was validated by qRT-PCR. We have also predicted the potential target genes of the abundant known and novel miRNAs, and subjected them to GO and pathway analysis, revealing the involvement of miRNAs in many important biological phenomenon including meiosis and p53-related pathways that are implicated in the regulation of spermatogenesis. Conclusions: This study reports the first genome-wide miRNA profiles in human testis using a NGS approach. The presence of large number of miRNAs and the nature of their target genes suggested that miRNAs play important roles in spermatogenesis. Here we provide a useful resource for further elucidation of the regulatory role of miRNAs and piRNAs in the spermatogenesis. It may also facilitate the development of prophylactic strategies for male infertility. - Funding: This work was supported by the National Basic Research Program (2013CB947900 and 2012CB944402) of China (973), and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-R-07). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. miRNAs are the class of endogenous non-coding RNAs, 19 to 25 nucleotides in size, which can regulate gene expression at either the transcriptional or post-transcriptional levels. Many studies have shown that miRNAs play an important role in various cellular processes, such as growth, proliferation, differentiation and death [1]. However, biological functions of many miRNAs are largely unknown, particularly in human spermatogenesis. Spermatogenesis is a complex process through which diploid germ cells proliferate and differentiate into haploid spermatozoa. Emerging evidence has shown that small RNAs are essential for spermatogenesis and male fertility [2,3]. First, several expression profile studies using cloning or microarray approaches have demonstrated that numerous miRNAs are exclusively or preferentially expressed in the testis or male germ cells of human and mouse [49]. Second, miRNA expression pattern appears to be different between immature and mature testes [7,10,11]. Last, spermatogenesis is disrupted at the early stage of proliferation and/or differentiation in mice with conditional knock-out of Drosha or Dicer [12]. Additionally, several studies have found that some miRNAs participate in mammalian spermatogenesis. For example, miR-122a is predominately expressed in post-meiotic male germ cells and promotes the degradation of transcripts of transition protein 2 (TNP2), a post-transcriptionally regulated testis-specific gene that involved in chromatin remodeling during mouse spermatogenesis [5]. miR-383 is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation by acting as a negative regulator of proliferation by targeting IRF-1 (Interferon regulatory factor 1) [13]. miR-372 and miR-373 can also promote the proliferation and tumorigenesis of primary human cells by neutralizing p53-mediated CDK inhibition, which possibly through the direct inhibition of tumorsuppressor LATS2s expression [14]. miR-184 whose expression was restricted to the germ cells from spermatogonia to round spermatids is involved in the post-transcriptional regulation of mRNAs of nuclear co-repressor 2 (ncor2) in mammalian spermatogenesis [15]. Recently, it has also been shown that miR-18, a member of the mir-1 cluster of miRNAs, directly targets heat shock factor 2 (hsf2), a transcription factor involved in spermatogenesis [16]. All these studies suggest that miRNAs are involved in the regulation of gene expression during spermatogenesis. PIWI proteins are the subset of the Argonaute proteins and expressed predominantly in the germline of various organisms. They are essential for germ cell maintenance and spermatogenesis in Drosophila and mammals [17]. MILI, MIWI, and MIWI2 are three mouse PIWI proteins that bind small RNAs of ,2431 nt termed as PIWI-interacting RNAs (piRNAs) [1822]. Recent studies suggested that piRNAs and PIWI proteins also play crucial roles in spermatogenesis. In Mili-deficient mice, spermatogenesis is arrested at the pachytene spermatocyte stage [23], while, Miwi2deficient mice display a defect in the early prophase of meiosis I and a marked and progressive loss of germ cells with age [24]. Previous studies indicating that miRNAs and piRNAs are required for normal spermatogenesis but detailed information on these RNAs in human testis are still not yet illustrated. A comprehensive profiling of testis-specific small RNAs will provide an insight into the mechanisms by which these small RNAs coordinate their target genes to regulate spermatogenesis and thus facilitate the understanding of causes for human male infertility. NGS has become increasingly popular in recent years because it can generate a massive amount of sequence data. It was shown to detect 25% more transcripts than microarray analysis [25]. NGS can successfully discover low abundance novel miRNAs due to its high sensitivity in detecting the genes with low expression levels than microarray [2630]. NGS has revolutionized many aspects of genetic and biomedical research [31]. In the present study, we de (...truncated)