The AML Prognostic Marker, AF1q, Is Directly Regulated by MiR-29b.

Abstract 2603

Poster Board II-579

We have consistently shown that elevated expression of AF1q, an MLL fusion partner, is a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetics (NC-AML), and adult myelodysplastic syndrome (MDS). However, the underlying mechanism of how AF1q is regulated in normal and abnormal hematopoiesis is still unclear. Our previous studies suggest that AF1q is highly regulated during hematopoietic cell differentiation and development and it is known that genes related to cell development and differentiation are likely to be regulated by various microRNAs. We used a variety of the web based programs to identify microRNA candidates that may potentially regulate AF1q based on the predicted targeting efficiency. We found the strongest predicted binder to the AF1q 3′untranslated region (3′UTR) was miR-29b, a member of the miR-29 family which has recently been characterized to regulate a member of the Bcl-2 family protein, Mcl-1 and other leukemia related oncogenes. We found that MiR-29b expression had a significantly inverse correlation (p<0.05) with AF1q expression in a cohort of more than 60 AML patients. This relationship is consistent with microRNA/AF1q regulation. In order to determine if miR-29b directly regulates AF1q, we chose H157 and SKMES1 lung cancer cell lines which were known to have high AF1q expression and high transfection efficiency to test if transfection of miR-29b into cells can suppress its endogenous AF1q expression. Our data showed that transfection of miR-29b into these two cell lines could indeed significantly suppress the AF1q expression both in H157 (p=0.001) and SKMES1 (p=0.004) cells. Then we wanted to determine if the miR-29b binding domain is specific in the AF1q 3′UTR region. Two reporter plasmids that contained the germline AF1q 3'UTR (GFP-A3U) and mutant AF1q 3′UTR in its microRNA binding site (GFP-A3U-Mutant) were constructed. We found that GFP readout was significantly suppressed in a stable transfection with native AF1q 3′UTR (GFP-A3U); in contrast, the GFP readout was not suppressed in a stable transfection with GFP-A3U-Mutant, suggesting that miR-29b is able to bind to the germline AF1q 3′UTR but not to the mutated 3′UTR (GFP-A3U-Mutant). These observations proved that miR-29b specifically regulates AF1q expression through its binding to the AF1q 3′UTR. Taking these observations together, we conclude that miR-29b directly regulates the leukemia associated gene AF1q. Elevated AF1q expression was found to have clinical significance in AML, thus, these findings warrant further investigation to determine if miR-29b will be able to serve as a prognostic biomarker for AML patients.