Expression Characterization Of MiR-34/277/279 And Their Roles On Expression Regulation Of Important Genes In Eyestalk Of Scylla Paramamosain

MicroRNAs(miRNAs)are a kind of non-coding RNA that are about 22 nucleotides.They play their functions in post-transcriptional regulation of gene expression.miRNAs pair with partially complementary sites in the 3? untranslated regions(UTRs)of target mRNAs,leading to translational repression and mRNA degradation of their target mRNAs.Animal miRNAs are able to recognize their target mRNAs by using as little as 6–8 nucleotides(the seed region)at the5' end of the miRNA,which is not enough pairing to induce cleavage of the target mRNAs.At present,although a lot of miRNAs have been studied in many species,the function of the miRNA in the regulation of development and reproduction in Scylla paramamosain has not been reported.Eyestalk is an important regulating organ in the process of development and reproduction in crustaceans containing molt-inhibiting hormone(MIH),crustacean hyperglycemic hormone(CHH),ecdysone receptor(EcR),farnesoicacid O-methyltransferase(FAMeT),red pigment aggregation hormone(RPCH)and so on.Studying the regulation role of miRNAs in eyestalk will contribute to illustrate the expression characterization of genes and the interaction between genes.Furthermore,studies of expression patterns of miRNA-34/277/279 at different stages of embryogenesis and ovarian development will reveal the expression characteristics of these genes,and to clarify the regulation mechanisms of eyestalk in regulation of reproduction in the crab.The detailed results are listed as follows:1)Bioinformatic analysis suggested that there are a putative binding site for miR-34 in the3?-UTR of MIH,CHH,EcR,FAMeT,and RPCH genes;a putative binding site for miR-277 in the 3?-UTR of VIH and a putative binding site for miR-279 in the 3?-UTR of MIH.Luciferase reporter assay showed that the miR-34 negatively regulates the expression of MIH,CHH,EcR,FAMeT,and RPCH genes;the miR-277 negatively regulates the expression of VIH gene,and the miR-279 negatively regulates the expression of MIH gene.2)The expression level of the miRNA-34 at early embryonic development stages(from cleavage stage to 5 pairs of appendages stage)was low.The expressionof the miRNA-34 was significantly increased at the stage of 7 pairs of appendages.The expression of the miRNA-34 achieved a small peak in eye-pigment stage I and II,then the expression increased significantlyat pre-hatching stage.Among different developmental stages of ovarian,the expression of miR-34 had no significant difference in the eyestalk at every stage;miR-34 was higher at early stage than at middle stage and late stage in the ovary in ovarian development;miR-34 was significantly increased at late stage in the cerebral ganglion in ovarian development;miR-34 had high expression but no significant difference in hepatopancreas at each stage of the ovarian development;In thoracic ganglia,the expression of miR-34 was higher at every stage and rose along with ovarian developing.3)Similar to miR-34,the expression level of miR-277 at early embryonic development stage(from cleavage stage to 5 pairs of appendages stage)was low.The expression of the miR-277 was significantly increased at the stage of 7 pairs of appendages.It achieved a small peak at eye-pigment stage I and II,then increased significantly at pre-hatching stage.In ovarian development,in the eye stalk,the expression of miR-277 had no significant difference at every stage;In the ovary,the expression of miR-277 was higher at early stage and then reduced with ovarian development;In the cerebral ganglion and hepatopancreas,the expression of the miR-277 was higher at early stage and late stage than at middle stage;In thoracic ganglia,the expression of miR-277 was higher at late stage than at early and middle stage.4)The expression of the miR-279 presented at low level and there was no significant difference before stage of 7 pairs of appendages in the process of embryonic development;From7 pairs of appendages stage,the expression of miR-279 began to rise,and achieved a peak at eye-pigment stage I,and then reduced quickly until hatching stage.In ovarian development,the expression of miR-279 had no significant difference in the eyestalk and ovary;In the cerebral ganglion and thoracic ganglia,the expression of miR-279 was high at every stage in ovarian development and higher significantly at late stage than at early and middle stage;In the hepatopancreas,the expression of miRNA-279 was low at every stage but was higher at the early stage.