ISOLATION AND CLONING OF Echinococcus granulosus microRNAs

CUCHER M; ROSENZVIT MC

Congreso; XXIII International Hydatidology Congress; 2009

ISOLATION AND CLONING OF Echinococcus granulosus microRNAs Cucher M, Rosenzvit M. Depto. de Microbiología, Facultad de Medicina, U.B.A. Buenos Aires, Argentina. marcecucher@gmail.com, TE: (0054)-11-5950-9500, ext. 2192. Introduction: microRNAs are non-coding small RNAs that negatively regulate the expression of their target mRNAs. Their importance in many biological processes has been widely documented since their discovery, mainly in development. Our aim was to prove the existence of this regulatory mechanism in cestode parasites, especially in Echinococcus spp. Materials and Methods: Protoscoleces were obtained from porcine hydatid cysts. Up to 200-nt-in-length RNA was isolated, gel size-fractionated, concatemerized after 5´and 3´-linkers ligation and used to transform Escherichia coli competent cells. Plasmid sequencing was performed and bioinformatic analyses were carried out using E. multilocularis genome as reference. Results: More than 1000 clones were obatined, each with a different concatemer insert. So far, more than 100 clones were sequenced yielding 196 sequences, 22.5% of which corresponded to rRNA, 2.5% to ESTs, 29.6% to known microRNAs, 13.3% to putative new microRNAs and the remaining 32.1% of the sequences could not be classified yet. Among the conserved microRNAs, some of developmental proved importance in other organisms were cloned, such as miR-125 and miR-10. Regarding the putative new ones, the bioinformatic analyses showed that they present microRNA features, i.e. length distribution (20-23 nt) and predicted secondary hairpin structure. In addition, many of them have been cloned several times and show variability in their 3´end, facts that reinforce their microRNA nature. Discussion: Unravelling the mechanisms of development regulation in parasites is of main importance in order to develop strategies to control them. The results obtained in this work constitute the basis of future studies on E. granulosus differentiation regulation, either between stages or strains, and eventually their extrapolation to other organisms. Keywords: Echinococcus granulosus, microRNAs, development.