MicroRNA-181a* targets nanog in a subpopulation of CD34+ cells isolated from peripheral blood

Abstract

Background and Objectives: Hematopoietic stem cells (HSC) are the most widely studied and characterized adult stem cells, which play an essential role in sustaining the formation of blood and immune system. The ease of their manipulation, the lack of serious ethical issues, and, in the autologous setting, the absence of their immunogenicity, have made them an attractive tool for developing stem cell-based therapies. Exploiting the properties of HSC by microRNA (miRNA) profiling offers an attractive approach to identify new regulators of stem cell fate. Although numerous miRNA have been screened from hematopoietic stem cells (HSC), the targets corresponding to many of these miRNA have not yet been fully elucidated. Therefore the objective of this study is to generate a miRNA profile from a subpopulation of adherent CD34+ HSC isolated from G- colony-stimulating factor mobilized peripheral blood aiming to understand the role of selected miRNA in regulating HSC stemness. Methods: CD34+ cells from patients' blood were isolated using a CD34+ isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. miRNA profiling of adherent and nonadherent CD34+ cells was done using TaqMan Array MicroRNA Cards. Nanog expression levels was tested using a dual-luciferase reporter construct for miR-181a* or its mutant variant and Nanog 3′ UTR mRNA. Results: In this study, we have identified eight clusters of miRNA that were differentially expressed in an adherent subpopulation of CD34+ stem cells. Further analysis of one of the clusters by bioinformatics revealed that a miRNA, miR-181a*, which is highly expressed in the adherent CD34+ cells, affects the expression levels of Nanog, a stem cell surrogate marker. We show specifically by reporter assay and mutational analysis that miR-181a* targets a seedless 3′ compensatory site in the 3′UTR of Nanog and affects gene expression. We demonstrate that inhibiting miR-181a* upregulates the Nanog expression level, in addition to an increase in alkaline phosphatase activity. Conclusions: In conclusion, our results highlight a new stem cell-related target for the miR-181 family and show that miR-181a* directly targets Nanog in a subpopulation of CD34+ stem cells suggesting a possible role of miR-181a* in regulation of adherent CD34+ HSC cells stemness.