| MicroRNA (miRNA) is a novel class of endogenous non-coding post-transcriptional regulatory small RNA, by interacting with target mRNA in the 3′-UTR, inhibiting translation of target mRNA or cause its degradation, and playing a important regulatory role of target gene expression. Recent studies showed that miRNA regulate 30% of the protein coding genes, and the identification and functional analysis of miRNA have become hot topic in molecular biology. Presently, it has been identified in different species and the function of miRNA has been well, involving development, proliferation and differentiation, apoptosis, insulin secretion, fat metabolism and even cancer including almost all biological processes.Pig is not only important agricultural economic animals, but also a good model organism of growth, development, reproduction and other physiological processes. However, the research on pig miRNA lags far behind in the human, mouse and other species (only 74 mature miRNAs in miRbase13.0), and even less than cows, chickens and other poultry species. The main reason is that it is difficult to fully identify the pig miRNA by the conventional method of cloning and bioinformatics. However, the second generation high-throughput sequencing technology provides a powerful tool for the identification of porcine miRNA. In addition, the pig miRNA research is focused on the muscle and fat tissue, while the tissues of reproductive traits are very little.In the present study, the identification and differentially expressed analysis of miRNAs by use of the Solexa sequencing technology combined with bioinformatics analysis were based on the construction of immature and mature pig testis small RNA cDNA libraries. We then predicted the target genes of differentially expressed miRNAs by the use of biological information software and analyzed the function of target genes. Further, analyzed the expression of some differentially expressed miRNAs in different stages of testicular tissue and various tissues, the results show that: 1. Deep sequencing of the two libraries by using Solexa sequencing technology successfully generated 14,802,526 and 14,660,953 unfiltered raw reads, of which 12,738,518 and 12,660,584 were high-quality greater than 18 nt small RNA sequences, corresponding to 1,336,183 and 2, 053, 109 unique sequences, respectively.2. In the immature library, the vast majority of small RNAs were 21-23 nt in length (> 60%), with 22-nt small RNA being the most abundant. In the mature library, however, we found a distinct bimodal distribution from the length distribution and abundance of small RNAs, with a peak around 21-23 nt and another distinct peak around 26-30 nt. Overall, the peak represented by the 26-30nt size class (67%) was greater than that for the 21-23nt-size class (11.5%). This suggested that the small RNA are significant difference in the overall composition and more abundant in the the mature pig testis.3. Bioinformatics analysis identified a total of 398 porcine miRNA of known and homologous conserved and 138 new pig miRNA candidates, including 18 new pig-specific miRNAs.4. we found that some special phenomenon regarding the known mature miRNA sequence that are worthy of further exploration, such as the abundance of certain miRNA * sequence is much higher than the corresponding mature miRNA; miRNA sequence length and sequence heterogeneity at the 5′and 3′ends; the abundance of 38 isoforms sequence is much higher than the mature miRNA registered in the miRBase; 12 specific miRNA that part of the sequence derived from miRNA precursor loop region or the remote of the 5′and 3′arm.5. 122 known and conserved miRNAs were differentially expressed between the immature and mature testis, of which 96 were significantly up-regulated and 26 were significantly down-regulated. The real time qRT-PCR analysis of ten miRNAs verified the result of Solexa sequencing.6. We predicted 2,252 target genes for the nineteen most significant differentially expressed miRNAs corresponding to 3,398 interaction sites. GO and KEGG Pathway analysis showed that 112 target genes associated with the reproductive process, suggesting that these target genes of the differentially expressed miRNAs may be involved in the pig testis development or spermatogenesis.7. Real time qRT-PCR of the different developmental stages of testis results showed that the overall expression trend of seven miRNAs had a certain regularity: miR-9-1, miR-30a, miR-122a, miR-145 and miR-183 expression levels increased gradually with the increase in months; miR-196 and miR-221 expression levels decreased gradually; while the other three miRNAs, miR-323, miR-199b *, let-7c irregular expression.8. Real time qRT-PCR of the different tissues results showed that miR-122a with the highest expression in the liver and relatively strong expression in the testis of all developmental stages; miR-9-1, miR-183, miR-199b * and miR-323 were highly expression in some tissues; miR-30a, miR-145, miR-196, miR-221 and let-7c were co-expression in all tissues. Furthermore, miR-9-1 and miR-145 with the highest expression in the spleen; miR-30a and miR-221 with the highest expression in the kidney; miR-199b * and miR-323 with the highest expression in the heart; miR-196 with the highest expression in the stomach; let-7c expression in the testis at birth, followed by muscle; miR-183 expression was highest in the lungs, weaker expression spleen and in the liver.In this study, the miRNAomics analysis between the immature and mature porcine testicular tissue were performed, and the results show that miRNA may play an important role in the testis development and spermatogenesis, which laid the foundation for further study the regulatory role of porcine miRNA in spermatogenesis and the molecular mechanism of reproductive traits. |
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