Inhibition of the Transcription Factor RUNX1 Causes Glycosyltransferase a Repression

Background: The ABO blood group system is unequivocally the most important in clinical transfusion medicine. Furthermore ABO is implicated in the development of a number of human diseases. The ABO antigens are not confined to RBCs but are widely expressed in a variety of human cells and tissues. Thus, ABO matching is critical not only in blood transfusion but also in cell, tissue and organ transplantation. The molecular genetic basis of the ABO system has been known since 1990. However, despite extensive investigations about regulation of ABO blood group receptor expression, the mechanism is not fully resolved. Previously we found that miRNAs plays a critical role in regulation of ABO blood group antigen. Numerous miRNAs which were up- or downregulated in RBCs of blood group O and of heterozygous genotypes as compared to homozygous genotype possess potential binding sites in the 3'UTR of several transcription factors, such as SP1 and RUNX1. Here we show that silencing of the transcription factor RUNX1 leads to downregulation of blood group A antigen.

Methods: We performed knockdown experiments for RUNX1 by lentiviral gene transfer of shRNA in primary hematopoietic stem cells (HSCs) and analyzed blood group A-antigen expression using different method, including flow cytometry, western blot and qPCR.

Result: Knockdown of RUNX1 in HSCs leads to a 10-20% reduction of blood group A positive erythroid cells and a 30-40% reduction of blood group A antigens per cell in differentiated RBCs. Furthermore, microarray analysis showed a significant increase of miR-215-5p and miR-192-5p in RBCs of blood group O as compared to homozygous genotype. RUNX1 is known to be a target gene for these miRNAs.

Conclusion: Glycosyltransferase A and B expression is regulated by different miRNAs, via simultaneously targeting of the transcription factors SP1 and Runx1 and glycosyltransferase A and B mRNA. The knowledge of the role of microRNAs and the transcription factors SP1 and RUNX1 in the expression of blood group antigens may be extended to other blood groups (Rhesus, Kell, Duffy) and may open the door for therapeutic interventions in diseases where blood group receptors promote disease pathology.