Cloning And Identification Of Micrornas From Giardia Canis

MicroRNAs（miRNAs）ranging from19~24nt is a kind of small non-codingRNAs. They play a very important role in cell proliferation, apoptosis and celldifferentiation. Generally, miRNAs interact with their targets by completecomplementary base pairing or partly complementary base pairing to induce mRNAsdegradation or translational inhibition and lead to gene translational silencing.Now, there is only a limited understanding about the molecular biologycharacters of Giardia lamblia, especially in the aspect of the mechanism of generegulation. Giardia mRNA has an extraordinary short5’UTR(0-14nt) and3’UTR(10-30nt) eliminating the chance of transcriptional regulation in Giardia. Anotherimportant gene regulation mechanism is called translational regulation, one of whichis miRNAs-induced gene silencing. Until now, two critical proteins Dicer and Agowhich are prerequisite in the process of miRNAs biogenesis and function, respectively,are found in Giardia indicating the presence of miRNAs involved gene regulation inGiardia. What’s more, six functional Giardia miRNAs were identified and testedrecently. They are miR2, miR3,miR4, miR5，miR6and miR10. miR3regulates theexpression of histone H2A, and there are no precise targets for miR5in spite of the21predicted potential targets. While, miR2, miR4, miR6and miR10all regulate theexpression of VSPs manifesting that miRNAs may play an important role in immuneevasion and other cell process.In order obtain enough information of Giardia miRNAs, in this study,we aimedto identify more miRNAs using Giardia canis. Firstly, we constructed the GiardiacDNAs pool using tail-adding reverse transcription, and got74clones containingshort sequences(19-24nt); Secondly,14sequences located in the transcriptional regionwere selected as the primary candidates, and then3candidate sequences were selected for being able to form a stem-loop structure with its upstream and downstreamsequence. After that, we identified the candidates by stem-loop Real-time PCR, DSLE,primer extension and Dicer cleavage assay in vitro.At last, we identified twomiRNAs: GmiR-6and GmiR-9. MiRNAs functional assy indicating that the twomiRNAs can function as other miRNAs by gene silencing with GmiR-6supressingthe target gene expression at about86.5%and GmiR-9at about92.7%. Finally, weutilized miRNA target prediction software: miRanda to find potential miRNAs targetsin Giardia virus genome. And we found there is no potential targets for GmiR-9inGiardia virus genome, while there are two potential targets for GmiR-6in Giardiavirus genome indicating that miRNAs may play an important role in the interaction ofGiardia and Giardia virus. This layed the groundwork for the study of the regulationmechanism and functions of Giardia miRNAs.