Isolation and cloning of Echinococcus granulosus microRNAs

CUCHER M; ROSENZVIT M

Congreso; XXIII International Congress of Hydatidology; 2009

Introduction: microRNAs are non-coding small RNAs that negatively regulate the expression of their mRNA targets. Their importance in many biological processes and organisms has been widely implied since their discovery, with strong emphasis in development. The aim of this work was to prove the existence of this regulatory mechanism in cestode parasites, especially in the genus Echinococcus sp. Materials and Methods: Protoscoleces were obtained from porcine hydatid cysts within the first 24 hours of slaughtering. Samples showing more than 95% viability were washed several times with PBS and stored in liquid nitrogen until their use. Up to 200-nt-in-length RNA was isolated, gel sized fractioned to isolate fragments between 19-40 nt and concatemered after 5´and 3´-linkers ligation. Escherichia coli competent cells were transformed with the concatemers and plasmid sequencing was performed. Bioinformatic analysis was performed 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, 21.4% of which corresponded to rRNA, 2.5% to ESTs, 29.6% to known microRNAs, 13.3% to putative new microRNAs and the remaining 33.2% of the sequences could not be classified yet. Among the conserved microRNAs, some of developmental probed importance in other organisms were cloned, such as miR-125, miR-10. Regarding the putative new ones, the bioinformatic analyses showed that they present microRNA features, such as length distribution (20-23 nt) and predicted secondary hairpin structure. In addition, many of them have been cloned several times and show certain degree of 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 may constitute the basis of future studies on E. granulosus differentiation regulation, either between stages or strains, and eventually their extrapolation to other organisms.