Identification And Characterization Of MicroRNAs In Antler Cartilage And Velvet Of Red Deer(Cervus Elaphus) At Rapid Growth Stage By Deep Sequencing

Deer antlers are the only mammalian appendages capable of full renewal, and therefore offer a unique model to explore how nature has solved the problem of epimorphic regeneration in mammals. Antler regeneration takes place in yearly cycles from its pedicle, a permanent protuberance on the frontal bone. Antler growth has obvious seasonal and late spring to early summer is the rapid growth stage, up to 2cm/d. Histologically, growing antlers consist of internal (cartilage and bone) and external (velvet, blood vessels, and nerves) components. It has reported that cartilage and velvet both play important roles in antler regeneration. However, mechanisms underlying how cartilage and velvet work together and grow fast on rapid growth stage is thus far unknown. microRNAs (miRNAs), a kind of endogenous, single stranded, about 22 nucleotides non-coding small RNAs, are hotspots recently. They function at post-transcription level via base-pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules are silenced by the following processes: cleavage of the mRNA strand into pieces or/and less efficient translation of the mRNA into proteins. It has been confirmed that miRNAs function widely among animals, plants and virus. Linked miRNAs and antler regeneration together, so we asked whether antlers express consensus and novel miRNAs? What different expression levels of miRNAs between different tissues of antler at certain growth stage?This study collected antlers of red deer growing for 60 days. Cartilage and velvet were isolated and small RNA libraries used for deep sequencing were established, respectively. Then data was further mined with bioinformatics methods. Studies in this study included:(1) small miRNA deep sequencing; (2) miRNA sequences analyzing and identifying conserved and novel miRNAs; (3) comparing different expression levels of miRNAs between antler cartilage and velvet; (4) predicting secondary structures and putative target genes of miRNAs and performing annotations for target genes; (5) real-time PCR validating deep sequencing results. Results showed:1. Through deep sequencing, antler cartilage and velvet generated 13,513,502 and 5,524,073 raw reads, respectively. After filtering low quality reads,9,520,645 and 3,621,894 mappable reads were left and 389 and 295 unique miRNAs identified in the tow libraries.2. miRNAs are about 22-25 nucleotides (nt). In cartilage library, the most small RNAs were 18nt (31.5%), while 22nt (18.0%) in velvet library.3. It identified 611 conserved and 73 novel miRNAs that classified into 7 categories.4. There were many isomiRNAs among conserved miRNAs. Terminal bases changes occurred in 262 miRNAs, in which miRNAs with bases insertion were more than deletion and changes happened at 3’end were more than 5’end. Besides,82 miRNAs had bases substitution in the sequences, including 58 miRNAs with transition and 28 miRNAs with transversion.5. Top expressed 20 miRNAs in cartilage and velvet libraries shared the most proportion, both exceeded 80%.6. Firstly identified antler miRNAs with homologous alignment. Compared 611 conserved antler miRNAs with other annotated mammalian miRNAs in miRBase (v18.0) database. The results showed most miRNAs (422) were conserved between deer and cattle, then wild boar (42), mouse (37), sheep (35) and human (27).7. All identified miRNAs could form conventional stem-loop hairpin structures predicted by using mfold software. Putative target genes of conversed and novel miRNAs were predicted by using TargetScan software. The function of target genes needs further study.8. Fourteen miRNAs with different copy numbers were chosen to validate their expression levels by real-time PCR (RT-PCR). The results showed that data from RT-PCR was almost consistent with deep sequencing. So antler miRNAs identified from deep sequencing were believable.This study was on the perspective of the miRNAs working in antler regeneration. miRNA profilings of cartilage and velvet tissues were generated through high-throughput sequencing technology. After analyzing small RNAs sequence features, miRNAs showed different expression levels in different tissues. Screening miRNAs related to antler regeneration is pressing. This study provided references for further research on miRNAs biological functions and regulation mechanism in antler regeneration. It would place us in a better position to promote organ regeneration in mammals.