Analyzing And Identification Of Porcine Embryonic MicroRNA By Solexa Sequencing Approach

MicroRNA (miRNA) are a class of small, non-coding RNAs of~22nucleotidesin length that regulate gene expression by completely or incompletely binding to the3′-untranslated regions (UTR) of target mRNAs with degradation mRNA orinhabiting translation. It is now clear that miRNAs are involved in many physiologicaland biochemical processes, including proliferation, differentiation, and regulation ofgene expression during early embryonic development. The miRBase16.0(2010)shows that there are175,673,408, and1,048annotated miRNAs for Caenorhabditiselegans, Mus musculus, Rattus norvegicus, and Homo sapiens, respectively. However,there are only211miRNAs described for Sus scrofa. In particular, the full expressionpatterns of miRNAs regulating gene expression are still poorly understood inembryonic development.Therefore, based on the construction of head region and organ region small RNAlibraries, we used the Solexa sequencing technology to identify the expression ofembryonic miRNAs by the researching embryo day33of pigs (E33). Meanwhile, wepredicted the target genes of abundant miRNAs in cDNA libraries by using ofbiological information software and preliminary study functions of them, thenanalyzing the different expression of the highly expressed miRNAs in head, organregions and adult tissues. The results show that:(1) After Solexa sequencing, the twolibraries of head and organ were successfully founded including11,617,079and11,458,732raw reads, in which11,400,040and11,256,616were high-quality smallRNA sequences, respectively.(2) Bioinformatics analysis of the distinct miRNAsidentified,75previously known miRNAs and194candidate miRNAs were identifiedin head library,76known miRNAs and130predicted candidate miRNAs wereidentified in organ library. In addition,20miRNAs were verified to express in bothhead and organ regions by Poly(A)-tailed PCR.(3)50known miRNAs were differentially expressed between head and organ libraries, in which31miRNAs inhead library were significantly higher than these in organ library,19miRNAs in organlibrary were significantly higher than these in head library and22miRNAs nodifferences.(4) The expression of10abundant miRNAs in head and organ regionwere analyzed by qRT-PCR, miR-9expression levels was highest in head region andmiR-1expression levels was highest in organ region. Moreover, the expression ofmiR-1, miR-103, and miR-140*were highest in muscle, brain, and lung, respectively.(5) Furthermore, we performed additional investigation for identifying the potentialtarget mRNAs of10highly expressed miRNAs using PicTar and TargetScan, finallyselecting total50target genes. KEGG Pathway analysis showed that the50targetgenes may be involved in the pig embryonic developmental formation of nervoussystem, limbs, anterior-posterior and dorsal-ventral pattern.In this study, we successfully identified miRNAs of embryo day33of pigs andpredicted target genes of partial miRNAs, analyzing function of target genes. Ourresults provide valuable information for investigators interested in the regulation ofembryonic development in pigs and other animals and lay the foundation for furtherinvestigation the action and molecular mechanisms of miRNAs in pig embryonicdevelopment.