Characterization of miR-10a mediated gene regulation in avian splenocytes

Abstract

It is well established that microRNAs (miRNAs) are an important class of post-transcriptional regulators of gene expression. Although numerous miRNA expression profiles have been generated for many eukaryotic organisms, little is known about the specific functions of individual miRNAs in regulating gene expression. We previously reported that the miRNA, miR-10a, is highly expressed during spleen development in embryonic chicks. In this current study we have identified genes and potential pathways that are both directly and indirectly influenced by miR-10a expression. To achieve this goal, miRNA Real-Time (RT) PCR analysis was first utilized to examine miR-10a expression across tissues during both embryonic and post-hatch chick development. Next, microarray analysis was employed to determine alterations in global gene expression associated with miR-10a in embryonic chick splenocytes subjected to an in vitro miR-10a inhibitor treatment. Finally the miRNA target prediction algorithm miRanda was used to predict potential chicken genes directly targeted by miR-10a. A select group of potential miR-10a target genes was validated using an RCAS-miRNA expression based luciferase assay. Our results indicate that miR-10a is highly expressed in the avian spleen, lung, kidneys, and fat tissues. Functional analysis suggests that miR-10a is involved in regulating gene expression in pathways associated with Ras signaling, intracellular trafficking, and development of immune functions. Additionally, we confirmed that chicken HOXA1 is a miR-10a target gene, suggesting a conserved role for miR-10a in the regulation of hematopoiesis across vertebrates.