Mircrorna-550 Functions As a Critical Tumor Suppressor in Acute Myeloid Leukemia

Background:

Acute Myeloid leukemia (AML) is one of the most common and fatal form of hematologic malignancies. Recurring chromosomal aberrations and gene mutations have been shown to contribute to AML pathogenesis and clinical outcomes. However, no effective therapy is available to selectively target the cytogenetic and molecular abnormalities, except for PML-RARA in acute promyelocytic leukemia (APL) which can be targeted by all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). As a result, the majority of AML patients are suffering from unsatisfied treatment of standard chemotherapy, associated with high rate of relapse and inferior survival. Thus, better understanding of the molecular mechanisms underlying the pathogenesis and drug resistance of AML, and more effective treatments based on such understanding, are urgently needed.

MiRNAs are a class of non-coding RNAs which post-transcriptionally regulate targeted gene expression. They usually consist of 20~24 nucleotides. MiRNAs are closely involved in almost all physiological and pathological processes. The widespread dysregulation of miRNA expressions have been shown to be correlated with various types of malignancies, including AML.

In our recent publication, we show through Exiqon miRNA microarray analysis that several miRNAs are significantly down-regulated in most subtypes of de novo AML; miR-550 is one of them (Jiang et al., Cancer Cell, 2012). However, its role and regulatory mechanism in AML have been poorly elucidated. The present study is to investigate the biological functions and molecular mechanisms of miR-550 in AML.

Methods:

The expression levels of miR-550 were analyzed in multiple AML cell lines, AML patients' bone marrow (BM) mononuclear cells (MNC) and normal MNC control samples by using Taqman miRNA assay qPCR kit. Cell viability and proliferation assays, i.e., MTT assays, were performed in human AML cell lines with stable ectopic expression of miR-550 or control plasmids induced by retrovirus. Cell apoptosis and cell cycle were assessed via flow cytometry analysis. To determine the influence of miR-550 on the transformation capacity of mouse BM progenitor cells transduced with leukemic fusion genes, e.g. MLL-AF9 and AML-ETO9a (AE9a), colony-forming/replating assay (CFA) was carried out. To evaluate the effect of restoration of miR-550 expression/function in AML progression in vivo, we retrovirally infected leukemic blast cells carrying MLL-AF9 with miR-550 or empty vector, and performed secondary BM transplantation by i.v. injecting recipient mice with these donor cells. To identify potential target genes of miR-550, two independent AML patient datasets were analyzed and the correlation patterns between miR-550 and the candidate targets were shown.

Results:

Consistent with the results of our previous miRNA array, the expression level of miR-550 was significantly down-regulated in most AML patient samples and AML cell lines as compared with normal controls. In AML cell lines, retrovirus induced enforced expression of miR-550 resulted in G1-phase arrest, increased apoptosis, and inhibited cell growth and viability. In mouse BM progenitor cells, forced expression of miR-550 dramatically attenuated colony-forming capacity driven by MLL-AF9 or AE9a. Overexpression of miR-550 significantly inhibited progression of AML induced by MLL-AF9 (MLL-AF9+miR-550, with medium survival of 33 days; MLL-AF9, with medium survival of 27 days; P=0.01) in vivo.

We further analyzed in two independent AML patient datasets the expressional correlation between miR-550 and its potential gene targets predicted by miRanda, miRWalk, PITA and Targetscan, etc. 77 candidate target genes inversely correlated with miR-550 in expression. Amongst these genes, FOXE1, IGFBP5 and KSR2 etc. have been shown to be oncogenes and are closely related with AML pathology. Therefore, these genes are candidate oncogenic targets that we will focus on in the future.

Conclusions:

The above results suggest that miR-550 is an important tumor suppressor in AML. Through targeting a series of oncogenes, miR-550 represses the viability and proliferation of leukemic cells, promotes apoptosis and differentiation, and inhibits cell transformation. Our study indicates that down-regulation of miR-550 likely plays a critical role in AML pathogenesis and restoration of miR-550 might hold great potential in treating AML in the future.