To elucidate the complex physiological process of testis development and spermatogenesis in Sika deer, this study evaluated the changes of miRNA and mRNA profiles in the four developmental stages of testis in the juvenile (1-year-old), adolescence (3-year-old), adult (5-year-old), and aged (10-year-old) stages. The results showed that a total of 198 mature, 66 novel miRNAs, and 23,558 differentially expressed (DE) unigenes were obtained; 14,918 (8,413 up and 6,505 down), 4,988 (2,453 up and 2,535 down), and 5,681 (2,929 up and 2,752 down) DE unigenes, as well as 88 (43 up and 45 down), 102 (44 up and 58 down), and 54 (18 up and 36 down) DE miRNAs were identified in 3- vs. 1-, 5- vs. 3-, and 10- vs. 5-year-old testes, respectively. By integrating miRNA and mRNA expression profiles, we predicted 10,790 mRNA–mRNA and 69,883 miRNA–mRNA interaction sites. The target genes were enriched by GO and KEGG pathways to obtain DE mRNA (IGF1R, ALKBH5, Piwil, HIF1A, BRDT, etc.) and DE miRNA (miR-140, miR-145, miR-7, miR-26a, etc.), which play an important role in testis development and spermatogenesis. The data show that DE miRNAs could regulate testis developmental and spermatogenesis through signaling pathways, including the MAPK signaling pathway, p53 signaling pathway, PI3K-Akt signaling pathway, Hippo signaling pathway, etc. miR-140 was confirmed to directly target mutant IGF1R-3′UTR by the Luciferase reporter assays. This study provides a useful resource for future studies on the role of miRNA regulation in testis development and spermatogenesis.

Due to the lack of high-quality Sika Deer (Cervus nippon) transcriptome and sRNAome across multiple organs or development stages, it is impossible to comprehensively analyze the mRNA and miRNA regulatory networks related to growth, development and immunity response. In this study, we used single molecule-real time sequencing (SMRT-seq) and Illumina sequencing methods to generate transcriptome and sRNAome from ten tissues and four age groups of Sika Deer to help us understand molecular characteristics and global miRNA expression profiles. The results showed that a total of 240,846 consensus transcripts were generated with an average length of 2,784 bp. 4,329 Transcription factors (TFs), 109,000 Simple Sequence Repeats (SSRs) and 18,987 Long non-coding RNAs (LncRNAs) were identified. Meanwhile, 306 known miRNAs and 143 novel miRNAs were obtained. A large number of miRNAs showed organ-specific and age-specific differential expression patterns. In particular, we found that the organ-specific miRNAs were enriched in the brain, some of which shared only between the brain and adrenal. These miRNAs were involved in maintaining specific functions within the brain and adrenal. By constructing miRNA96mRNA interaction networks associated with Sika Deer immunity, we found that miRNAs (miR-148a, miR-26a, miR-214, let-7b, etc.) and mRNAs (CD6, TRIM38, C3, CD163, etc.) might play an important role in the immune response of Sika Deer spleen. Together, our study generated an improved transcript annotation for Sika Deer by SMRT-seq and revealed the role of miRNA in regulating the growth, development and immunity response of Sika Deer.