| MicroRNA (miRNA) is a kind of endogenous non-coding small RNA which is about 22 nucleotide (nt), single chain and highly conserved. Detected widely in eukaryotic organisms, miRNA mainly target to the 3’untranslated regions of target gene’s mRNA, resulting in the translational inhibition or degradation of the target gene, which plays a very important role in the post-transcriptional regulation of target genes. As previous studies shown, numerous miRNAs are exclusively or preferentially expressed in the testis or male germ cells and miRNA expression pattern also appears to be different between immature and mature testes, which implies that the crucial role of miRNA in the development of germ cells and testes. Meanwhile, pig is an excellent model animal in the research of agricultural economy and medical field, and furthermore, high-throughput sequencing technology can rapidly, accurately and batch gain the miRNA profile. Thus, we selected high-throughput sequencing technology to analyze the profile of the microRNA expressed in immature and mature porcine testes, to explore the important role of miRNA in both testicular development and regulation of spermatogenesis, to reveal the regulation mechanism of miRNA regulating spermatogenesis and facilitate the development of prophylactic strategies for male infertility. Therefore, through high-throughput sequencing technology, this study constructed two different miRNA profile, namely immature and mature porcine testes respectively, which have analyzed and compared the characteristic both immature and mature porcine testes. The main results are shown as follow:1. using high-throughput sequencing technology, we effectively measured two small RNA libraries derived from immature (20-day-old) and mature (210-day-old) porcine testis samples, and yielded over 20 million high-quality reads in this study.2. In total, we detected 834 mature microRNAs (miRNAs) encoded by 609 precursor (pre)-miRNAs. In which 396 miRNAs (228 pre-miRNAs) belong to the subgroup of porcine known miRNAs. Besides,65 miRNAs (49 pre-miRNAs) belong to the subgroup of porcine conserved miRNAs. In addition,373 miRNAs (332 pre-miRNAs) belong to the subgroup of porcine novel/candidate miRNAs.3. The miRNA expression profile in two different phases of both immature and mature porcine testes, show a common characteristic that only a few types of miRNAs comprised the majority of sequences. The top 10 miRNAs with the highest abundance contributed 58.88% and 77.52% of the total read counts in immature and mature libraries, respectively.4. Among 461 porcine known/conserved miRNAs, in total,334 miRNAs were differentially expressed between immature and mature porcine testes, liking this pattern that there are 228 and 106 miRNA are highly expressed in immature and mature porcine testes respectively.5. In total, we identified 3118 target genes predicted from 20 highly abundant DE miRNAs, and the GO annotations indicated that these target genes were mainly enriched in regulation of biosynthesis process, signal transduction, and energy metabolism, such as "DNA transcription", "phosphate metabolic process", "chromatin binding", and "response to steroid hormone stimulus". The KEGG pathway analysis identified 54 enriched pathways for these highly abundant DE miRNAs, and the "pathways in cancer", "MAPK signaling pathway", "focal adhesion", "Wnt signaling pathway", "neurotrophin signaling pathway", and "GnRH signaling pathway" ranked top among the enriched pathways.6. Mapping these miRNAs and differentially expressed miRNAs to chromosomes, result show 55 miRNAs (11.93%,55 of 461), and 43 DE miRNAs (12.87%,43 of 334) were located on the X chromosome, respectively. Among these X-linked miRNAs, we screen out 18 differentially expressed miRNAs that there are 13 miRNAs highly enriched in immature porcine testes and the other 5 miRNAs highly enriched in mature porcine testes respectively. According to previous studies, these highly expressed X-linked miRNAs that differentially expressed between immature and mature porcine testes, are of significant importance in both testicular development and regulation of spermatogenesis.In sum, miRNAs are extensively involved in spermatogenesis and that unraveling miRNA functions in the testis will further our understanding of the regulatory mechanisms of mammalian spermatogenesis. |
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