Identification of a novel miRNA from the ovine ovary by a combinatorial approach of bioinformatics and experiments

Abstract

MicroRNAs (miRNAs) are a class of short endogenous, single-stranded, non-coding small RNA molecules, about 19–25 nucleotides in length that regulate gene expression at the translation level and influence many physiological process, such apoptosis, metabolism, signal transduction, and occurrence and development of diseases. In this study, we constructed a library from the ovine luteal phase ovary by using next-generation sequencing technology (Solexa high-throughput sequencing technique) and identified 267 novel miRNAs by bioinformatics. One of the novel miRNAs (ovis_aries_ovary-m0033_3p), which expressed in the sheep ovary and testis, was confirmed by real time PCR and northern blot. Ovis_aries_ovary-m0033_3p was 21 nucleotides in length and located on chromosome 12, and it had 100% similarity to hsa-miR-214-3p, mmu-miR-214-3p, dre-miR-214and ssc-miR-214. Meanwhile, the pre-miRNA was 82 nucleotides in length and had a standard hairpin stem-loop structure. From the consistency of the sequence and structure, we speculated that ovis_aries_ovary-m0033_3p had a function similar to hsa-miR-214-3p, which is involved in the fine regulation of cell survival, embryonic development, breeding activities and resistance to ovarian cancer, so we defined it as oar-miR-214-3p. These experimental results will enrich the miRNA database for ovis aries and provide the basis for researching the regulation mechanism of miRNA in relation to breeding activities of seasonal breeding animals.

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

“…In this study, a testis small RNA library was successful constructed by using Solexa high-throughput sequencing technology to identify small RNAs of ovine. From the library we found that up to 94% of the miRNAs reads in the ovine testis were 20-24 nt, and the result was consistent with the typical size of mature miRNAs and the findings of previous studies (Zhang et al, 2009;Chang et al, 2015).…”

“…These specimens were immediately frozen in liquid nitrogen. RNA extraction and detection were as same as Chang et al (2015).…”

“…Library construction, sequencing and analysis of sequence data were as same as Chang et al (2015) Quantitative RT-PCR of known and novel miRNAs: The all process of qRT-PCR was as same as Chang et al (2015) (Meenakshi et al, 2017). The primers of the miRNAs for qRT-PCR are shown in Table 1.…”

“…The all process of Northern blotting was as same as Chang et al (2015). The probe sequences of miRNAs for northern blot analysis are shown in Table 2.…”

“…MicroRNAs are a class of endogenous, non-coding, single-stranded functional RNA molecules, approximately 19-24 nucleotides (nt) in length, that are widely present in animals (Chang et al, 2015;Raja et al, 2016). Since the first miRNA (Lin-4) was discovered in 1993 (Bruce and Gary, 1993), more than 28,645 miRNAs have been identified in eukaryotes using various experimental approaches or computational predictions (http://www.mirbase.org/ index.shtml; 7.1.2014).…”