Identification and characterization of novel microRNA candidates from deep sequencing

In our previous study, we screened a candidate new microRNA (miRNA) based on the deep sequencing and bioinformatics analysis. In this paper, we evaluated the novel miRNA in the following experiment: 1) the secondary structure of the precursor of novel-miR has the characteristic of a stem-loop hairpin structure, and mature miRNA is far from loops and bulges. 2) we used BLAST (Basic Local Alignment Search Tool) to compare the novel-miR sequence to that found in the GenBank. Novel-miR sequence existed in Mus musculus, Drosophila grimshawi, Rattus norvegicus, Xenopus laevis, Spodoptera frugiperda, Papio anubis, Salmo salar and so on. Then multiple sequence alignment (MSA) showed that sequence from 5 to 11 bp and 13 to 17 bp exhibited 100% similarity, where there is significant sequence conservation. Novel-miR showed similarity in the seed region with the known miR-3675-3p, indicating that these miRNAs are likely to belong to the same family and thus may share common biology. 3) novel-miR from MCF-7 and MB-MDA-231 was validated by Northern blot and detected in the serum and tissue samples of BC patients, respectively, by real-time PCR. The data showed that novel-miR was downregulated in the BC cancerous tissues and serum of breast cancer patients (P<0.05). 4) transfection of novel-miR mimics into MCF-7 cell significantly inhibited cell growth detected by CCK-8 assay (P<0.05). 5) to identify the mRNA targets of novel-miR, we performed a computational screen for genes with novel-miR complementary sites in their 3'-UTR using several open access databases. In addition, we used the CapitalBio® Molecule Annotation System V3.0 to perform gene ontology (GO) analysis on the target genes of novel-miR and specific biological process categories were enriched. 7 genes (CUL3, KRAS, ETS1, MNT, CNTN3, CCNK and FOXO3) which have a high prediction score and are associated with cell proliferation, apoptosis and cell cycle were chosen. 3'-UTR luciferase report assay suggested that miR-BS1 negatively regulated CNTN3. In the conclusion, novel-miR, named miR-3675b, is a true, functional and novel miRNA. Candidate novel miRNA from deep sequencing which will be qualified as a "real" miRNA must be validated by a series of functional experiments.