The Mir-23a∼Mir-27a∼Mir-24 Cluster Acts as a Tumor Suppressor In Leukemias by Post-Transcriptional Regulation of 14-3-3 Proteins

Abstract 3145

We and others have found that microRNAs (miRs) play major roles in normal hematopoietic differentiation, evidenced by the discovery of a small set of hematopoietic stem-progenitor cell (HSPC)-expressed miRs (HE-miRs) highly expressed in normal human CD34+ cells, which post-transcriptionally regulate specific mRNAs involved in hematopoiesis. Microarray data indicated that 1 of these HE-miRs, miR-27a (transcribed as part of the miR-23a cluster consisting of 3 coordinately transcribed miRs: miR-23a, miR-27a, miR-24-2) is absent or expressed at much lower levels in several human acute leukemias, as compared to normal human HSPCs. We further investigated the expression of miR-27a in acute leukemias, and confirmed via qRT-PCR that there is ≥ 2-fold decreased miR-27a expression in 3 of 6 acute myeloid leukemia (AML), 4 of 5 B precursor acute lymphoid leukemia (ALL), and 3 of 3 T cell ALL cell lines tested (10 of 14 cell lines total). Additionally, 5 of 7 primary B precursor ALL and 5 of 5 primary T cell ALL samples had decreased miR-27a expression compared to normal HSPCs. Over-expression of miR-27a in K562 cells resulted in 4-fold decreased proliferation and 2-fold increased apoptosis compared to control cells. Similarly, over-expression of miR-27a in 3 other acute leukemia cell lines (TF1, HL60, and REH) yielded a significant increase in the percentage of AnnexinV+/7AAD- cells compared to control cells (TF1 = 21.3% vs. 12.0%; HL60 = 19% vs. 4.5%; REH = 21.5% vs. 4.7%). Members of the 14-3-3 protein family act as oncogenes and support cell survival by interacting with and negatively regulating pro-apoptotic proteins such as Bax and Bad. The 14-3-3β isoform (YWHAB) is a predicted miR-27a target gene, based on transcriptome analysis of normal HSPCs, and miR prediction algorithms revealed 2 predicted miR-27a binding sites in its 3`UTR. In addition, there are 5 predicted miR-24 binding sites in the 3`UTR and coding region of YWHAB, and 4 other 14-3-3 isoforms have multiple predicted miR-23a cluster member binding sites in their mRNA transcripts: 14-3-3θ (YWHAQ, 1 miR-27a site), 14-3-3γ (YWHAG, 2 miR-23a, 5 miR-27a and 3 miR-24 sites), 14-3-3ζ (YWHAZ, 3 miR-27a and 5 miR-24 sites), and 14-3-3ε (YWHAE, 1 miR-27a and 1 miR-24 site). The expression levels of the other 2 cluster members, miR-23a and miR-24, are also decreased in human acute leukemia cell lines and patent samples, similar to miR-27a expression in the same leukemia cases. We have confirmed via luciferase reporter binding assays and Western blot analyses that both miR-27a and miR-24 specifically regulate 14-3-3β protein expression levels, while miR-27a specifically regulates 14-3-3θ. Moreover, combined miR-27a and miR-24 over-expression decreased 14-3-3ε protein expression on Western blots. Since knockdown of 14-3-3 family proteins is established to result in apoptosis in hematopoietic cells including K562, post-transcriptional downregulation of 14-3-3 proteins by miR-27a, miR-23a and miR-24 contributes to the miR-27a-induced apoptosis in leukemias. Thus, miR-27a acts as a traditional tumor suppressor gene and members of the miR-23a cluster cooperate to regulate oncogenic 14-3-3 isoforms, and consequently, cell survival, in human acute leukemias.