Expression And Functional Analysis Of Micrornas During Metamorphosis Of Paralichthys Olivaceus

microRNAs (miRNAs) are endogenous, non-coding, small RNAs of 20~25 nt, and exist widely in diverse tissues and organs from nematode to drosophila and human. miRNAs play key roles in many diverse biological and cellular processes, including growth, development and disease in animal and plant. Japanese flounder undergo drastically post-embryonic metamorphosis, resulting in dramatic morphological changes such as translocation of the right eye to the left side of the body, elongation and subsequent shortening of crown-like larval fin, and rebuilding of tissues and organs. miRNAs play key roles in regulating biological process of animal development, however its expression and function in Japanese flounder have not been reported up to now. The aim of the present study was to examine the effects of miRNA on regulating flounder metamorphosis. Firstly, Solexa sequencing and bioinformatics were used to analyse sequence of small RNA, and identify flounder miRNA during flouder metamorphosis. Secondly, miRNA microarray was used to study the differential expression of miRNA at different matamorphic stages of larvae. On the basis of these studies, expression of miR-1, miR-133a, and miR-206a related to muscle development were studied for their function on flounder metamorphosis.Small RNA during flounder metamorphosis were sequencing initially by Solexa sequencing. We analyzed the length distribution of small RNAs, which are 14-28 nt long in the library, and found that most small RNA are 20~23 nt in length. In order to discover flounder miRNAs, our dataset were compared to known metazoan miRNAs in miRBase 13.0 by miRAlign. We identified 140 conserved miRNA, 133 miRNAs belonging to 84 families and 7 miRNAs that did not belong to any family, and 57 conserved miRNA*s which came from 57 duplex-like miRNA:miRNA* pairs in which the mature miRNAs and miRNA*s aligned to the 5'end or 3'end region, respectively. We obtained 20 miRNA precursors by PCR amplification, and predicted their secondary structures of these miRNA precursors using mfold, and validated tht all 20 sequences could be properly folded into the typical hairpin structure. Real-time PCR, Northern blot, and miRNA microarray were used to validate further the 197 flounder conserved miRNAs. Meanwhile, we analysed further phylogenetic evolution of these miRNAs, and found that 84 miRNA families and 7 miRNAs were sorted into 5 groups based on their phylogenetic distribution. Some miRNA families are present in mammals, birds, amphibians, fish, insects and nematodes; some families are only present in mammals, birds, amphibians and fish; and some families are vertebrate-specific. Although the 84 miRNA families are highly conserved throughout different animal taxa, there are pedigree specific sequence substitutions in most of these families, for instance, a base substitution of miR-21 between fish and mammals, birds, amphibians, and of mir-29a between flounder and other fish. Nucleotide substitutions in mature miRNAs might cause diversity of regulating gene expression.miRNA microarray was used to identify differential expression of miRNA at two different metamorphic stages (17 and 29 dph) of larvae. We identified 66 miRNAs which were expressed differentially at both 17 and 29 dph. We analysed these miRNAs and flound that they might play important roles in many biological changes during flounder metamorphosis, such as miR-25 related to pigmentation, a skin-specific miR-203, muscle-specific miRNAs (miR-1, miR-133, and miR-206), and miR-126 related to cardiovascular development. Further analysis of expression and function of the flounder miRNA could be helpful in explaining the complex genetic networks that control flounder metamorphosis.On the basis of data of microarray and Solexa sequencing, real-time PCR was used to analyse the expression of miR-1, miR-133a, and miR-206a in the different tissues of adult flounder and the metamorphic process of larvae. Our studies showed that in adult flounder, miR-1 and miR-133a are mainly expressed in the muscle and heart, and miR-206a is mainly expressed in the muscle. We detected expression of miR-1, miR-133a, and miR-206a during metamorphosis of NC (normal control), TH-treated and TU-treated larvae, the results showed that TH could significantly promote expression of miR-1 and miR-206a, and inhibit expression of miR-133a at peak and later stags of metamorphosis. To study further function of miRNAs, we predicted the targets of miR-1, miR-133a, and miR-206a by RNAhybrid, and obtained 13 candidate targers of miR-1, 8 candidate targers of miR-133a, and 16 candidate targers of miR-206a. So, we deduced that during flounder metamorphosis, TH promotes expression of miR-1 and miR-206a which induce differentiation of myoblasts, accelerates transformation of larval-type muscle to adult-type muscle, and then controls the process of flounder metamorphosis.MyoD, Myf5, and Myogenin are important myogenic regulatory factors in muscle development, and play key roles in specification, differentiation, and proliferation of myoblasts. To study function of MyoD, Myf5, and Myogenin in flounder metamorphosis, real-time PCR was used to analyse their expression. Our results showed that MyoD expression levels were much higher than of Myf5 and Myogenin, and were promoted by TH at early metamorphosis, but were inhibited by TU at late metamorphosis; Myf5 expression were higher than that of Myogenin, and were promoted significantly by TH during metamorphosis, but were inhibited obviously by TU at late metamorphosis; Myogenin expression were lower, and were inhibited by TH at late metamorphosis, but were obviously promoted by TU at late metamorphosis. These results indicated that MyoD, Myf5, and Myogenin were regulated by exogenous TH and TU, and play key roles in controling the flounder metamorphosis.