Abstract microRNAs (miRNAs) play vital roles in several biological processes, including apoptosis, by negatively regulating the expression of target genes. The molecular mechanisms of the key survival signal, Akt family, have been extensively investigated, but whether is regulated by miRNA remains unknown. In a recent study of neuroblastoma samples, we found the expression of miR-149 to be lower in the high-risk group than the low- and intermediate-risk groups. Upon transfection of Be2C, a neuroblastoma cell line and HeLa cells with miR-149 oligo, no changes in cell growth were observed. On the other hand, ectopic expression of miR-149*, the cognate miRNA of miR-149, inhibited the growth and induced apoptosis in these two cancer cell lines. By TargetScan screening, Akt1, the predominant Akt isoform in most tissue, E2F1, and b-Myb were predicted as possible target genes of miR-149*. In Be2C and HeLa cells transfected with miR-149* oligo, reduced expressions of Akt1, E2F1, and b-Myb at protein level were detected by Western blotting, and decreased mRNA levels of Akt1 and E2F1 were also noted by RT-qPCR. Using luciferase reporter assay, the target sites were verified in the 3′UTRs of Akt1 and E2F1, but not in b-Myb, suggesting that Akt1 and E2F1 were the direct target and b-Myb was an indirect target of miR-149*. Silencing of Akt1 or E2F1 expression also led to similar apoptotic changes as miR-149* transfection in these two cell lines, suggesting that the pro-apoptotic effects of miR-149* was exerted at least in part by repressing Akt1 and E2F1 expressions. Importantly, analysis of primary neuroblastoma samples (n=56) revealed a significant inverse correlation of miR-149* with E2F1 expressions (p=0.026). Furthermore in stage1 samples, the mean expression level of miR-149* was significantly higher than in stage 2/3 (p<0.05), and that of E2F1 appeared to be lower. The results provide further support of an important role of miiR-149* in regulation of E2F1, especially in stage1 neuroblastoma tumors. Interestingly, using the reporter assays, excess miR-149 introduced by transfection to simulated its preponderance in the in vivo condition, could not overcome the repressive function of miR-149* on the target genes. This implies that the pro-apoptotic function of miR-149* may not be dampened by its predominant cognate, miR-149, in vivo. Our findings not only provided the first evidence that Akt1 is a direct target of a miRNA but also demonstrated that miR-149* is a pro-apoptotic miRNA by repressing the expression of Akt1 and E2F1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2038.