Additionally, Real-time PCR has proven to 
be a simple and accurate method to identify and measure the expression levels of miRNAs. Using this approach, we validated the presence of 9 out of 10 selected novel miRNAs. As an initial step toward understanding the biological function of miRNAs in E. histolytica, we searched for miRNA targets among annotated protein-coding transcripts of E. histolytica. Applying a cutoff value of 218 kCal mol21, we predicted a total of 32 genes as miRNA targets. The putative target genes appear to be involved in various biological processes. However, since the E. histolytica genome is not fully annotated and a great proportion of E. histolytica protein-coding genes have no known function, it is difficult to draw a conclusion as to whether these miRNA targets have any functional bias. Our results suggest that miRNA-controlled mechanisms may be involved in human infection of E. histolytica. Some of the predicted target genes with annotated functions that we found are involved in gene regulation and signal transduction, such as Ras family GTPase. These proteins have been implicated in key pathogenic processes of E. histolytica. Some Ras and Rho GTPase effectors, particularly kinases such as the PAKs and members of the mitogen-activated protein kinase cascade, have also proven tractable as pharmacological targets in humans. However, the importance of Ras effectors and downstream kinases in E. histolytica pathogenesis has not yet been explored. The liver is also a prime target for amoeba infection. This organ contains a plentiful source of iron, which is essential for the growth of this parasite. Amoeba trophozoites are able to take up ferritin from the liver and internalize this protein via clathrin-coated vesicles. The capacity to use ferritin as an iron source may explain E. histolytica’s high pathogenic potential in the liver. Since clathrin is a protein required for receptor-mediated internalization of lipid and protein molecules, its regulation by miRNAs as potential target detected here could help to reduce the pathogenic functions of this parasite in the liver. Another predicted target, the TolA protein, is involved in the translocation of group A colicins. The colicins are bacterial proteins that are active against Escherichia coli and other related species. TolA is anchored to the cytoplasmic membrane by a single membrane-spanning segment near N-terminus, leaving most of the protein exposed to the periplasm. It is noteworthy that one miRNA regulates the unconventional myosin IB protein. The pathogenicity of E. histolytica includes its capacity to phagocyte human cells. Motility requires polarization of E. histolytica that involves protrusion of a pseudopod containing actin and associated proteins and whole-cell propulsion after contraction of the rear of the cell, where myosin II and F-actin are concentrated.