Sequencing And Analysis Of Exosomal MiRNA Transcriptome In 'Pigeon Milk'

Pigeons?Columba livia?are altricial,and the newly hatched squabs do not have independent living ability.Essential nutrients and energy are supplied by pigeon milk?PM?which secrets from adult pigeon crop.PM mainly consists of desquamation of lipid rich cornified epithelium,and carries out the similar founctions to mammalian milk.Exosomes encapsuling protein,lipid,m RNA and miRNA,have been proved to widely exist in breast milk.Particularly,the miRNAs in exosomes play an important role in the intercellular signal communication and immune function.However,there is dearth of scientific literature to address the exosome and the exosomal miRNA in PM.In this study we isolated exosomes from PM in five different lactation stages?1,5,10,15 and 20d?by using the ultracentrifugation.Then the atomic force microscope?AFM?and Western blot were applied to for further identification.Through Solexa deep sequencing and bioinformatics analysis,exosomal miRNAs in PM were indentifed and compared with those in mammalian milk.The results show as follows:1.With an approximate width of 150 nm and a height of 8 nm,the morphology of PM exosome is similar to mammalian milk.In addition,the results of Western blot showed that the exosome marker protein CD63 is present in PM exosome.2.301 mature miRNAs were identified in the seven libraries,which contained 248conserved miRNAs and 53 novel miRNAs.Hierarchical clustering analysis showed that the five lactation stages were divided into two categories:?1d,5d and 10d?,?15d and 20d?.The top ten expressed miRNAs over five lactation stages correspond to 25 kinds of miRNAs.Four miRNAs?cli-miR-21-5p,cli-miR-148a-3p,cli-miR-10a-5p and cli-miR-26a-5p?were shared by all five lactation stages.3.According to the annotation of Pathway Central Database?SABiosicences,MD,USA?and miRBase 21.0,there are 58 immune-related miRNAs(P<10^-37,?²-test)and 38growth-related miRNAs(P<10^-29,?²-test)presented and enriched in PM exosomal miRNA libraries.4.By comparing the expression abundance of exosomal miRNA between PM and mammalian milk,the results showed that there were a significant proportion of conserved miRNA shared between PM and mammalian milk,for instance miR-148a,miR-21and miR-26a.Besides,the expression of miRNA showed a relatively high correlation between them?average r=0.57?.5.Exosomal miRNAs expression pattern across the lactation period in PM were assessed by analysis of the sequencing data using the Short Time-series Expression Miner software?STEM?algorithm.Three significant model profiles?profiles 1,2 and 3?were generated.Go and KEGG analysis reveals the PM exosome miRNAs mainly are involved in embryonic organ development and growth of related pathways,including MAPK,Wnt and insulin pathways.The above results have showed that the PM contain exosome as well as exosomal miRNAs,and those miRNAs may be involved in the synthesis of PM and play important roles in the development of squabs and PM production.