Discovery of two novel miRNAs from the Ovis aries by a combinatorial approach of experiments and bioinformatics

n the study, we successful constructed a library from the ovine testis using next-generation sequencing technology, and identified 219 novel miRNAs by bioinformatics. Two of the novel miRNAs (ovis_aries_testis-m0052_5p and ovis_aries_testis-m0165_5p), which were expressed in the sheep testis and ovary, and were confirmed by real-time PCR and northern blotting. Ovis_aries_testis-m0052_5p was 23 nucleotides in length, was located on chromosome 15, and had 100% similarity to mmu-miR-34c-5p, hsa-miR-34c-5p, gga-miR-34c-5p, and cfa-miR-34c. Ovis_aries_testis-m0165_5p was 21 nucleotides in length, located on chromosome 5, and had 100% similarity to mmu-miR-145a-5p, hsa-miR-145-5p, ssc-miR-145-5p, and bta-miR-145. The pre-miRNAs for Ovis_aries_testis-m0052_5p and Ovis_aries_testis-m0165_5p were 75 and 81 nucleotides in length, and both had a standard hairpin stem-loop structure. From the consistency of the sequence and structure, we speculated that ovis_aries_testis-m0052_5p had a function similar to hsa-miR-34c-5p, mmu-miR-34c-5p, and ovis_aries_testis-m0165_5p had a function similar to hsa-miR-145-5p, which were involved in the fine regulation of cell survival, spermatozoon generate, breeding activities. Therefore, we defined the identified miRNAs as oar-miR-34c-5p and oar-miR-145-5p. The results will enrich the miRNA database, and provide the basis for research into the regulatory mechanisms of miRNA in relation to breeding activities.