(E) As expected, let-7aCtargeted genes such as and were decreased in CXCR4-shRNA-OCI3 cells. of immature myeloid cells in BM and peripheral blood (1, 2). The disease carries an extremely poor prognosis, and the principal cause Azasetron HCl of treatment failure is usually chemotherapy resistance (2, 3). Leukemic cells have been shown to hijack the homeostatic mechanisms of normal hematopoietic stem cells and take refuge within the BM niche (4, 5). This mechanism is pivotal to the survival of residual cells after chemotherapy and consequently contributes to disease relapse. In recent years, interrupting the connection between leukemic cells and the tumor microenvironment by targeting the stromal-derived factor 1/CXCR4 (SDF-1/CXCR4) axis has become a stylish therapeutic approach for AML. Our group as well as others have shown that culturing of AML cells with SDF-1 (also known as CXCL12) promotes their survival, whereas adding neutralizing CXCR4 antibodies, SDF-1 antibodies, or the CXCR4 inhibitor AMD3100 significantly decreases it. BM-derived mesenchymal stromal cells can also safeguard AML cells from chemotherapeutic drugCinduced apoptosis (6, 7). Moreover, weekly administration of anti-human CXCR4 antibody to mice previously engrafted with human AML cells prospects to a dramatic decrease of human AML cells in BM, blood, and spleen in a dose- and time-dependent manner (8, 9). However, the mechanisms involved in this SDF-1/CXCR4 axisCtargeting progress are not fully comprehended. microRNAs (miRNAs) are small noncoding regulatory RNAs approximately 18C25 nucleotides in length (10, 11). Each miRNA has the potential to target a large number of genes. The discovery of miRNAs in the early 1990s opened a new era in understanding transcriptional and posttranscriptional regulation of gene expression in biological processes BIRC3 (11C13). miRNAs are now known to play functions in almost all aspects of malignancy biology, including proliferation, apoptosis, invasion and metastasis, and angiogenesis (14C16). In recent years, functional and prognostic studies have confirmed that miRNAs plays an active role in hematologic malignancies, and some miRNAs have been proposed as prognostic markers and therapeutic targets in leukemia treatment. Calin et al. found that 65% of B cell chronic lymphocytic leukemia patients have deletions of chromosome 13q14, a locus that includes miR-15a and miR-16-1, which consequently present downregulated expression (17). Garzon et al. reported that miRNAs including miR-15a, miR-15b, miR-16-1, miR-223, miR-342, and miR-107 are upregulated, whereas miR-181b is usually downregulated, in acute promyelocytic leukemia (18). miR-155 was reported to be upregulated in patients with an internal tandem duplication of the gene (19). Schotte et al. showed that 14 miRNAs are upregulated (miR-128a, miR-142-3p, miR-142-5p, miR-150, miR-181a, miR-181b, miR-181c, miR-193a, miR-196b, miR-30e-5p, miR-34b, miR-365, miR-582, and miR-708), and 5 downregulated (miR-100, miR-125b, miR-151-5p, miR-99a, and let-7e), in acute lymphoblastic leukemia cells compared with normal CD34+ cells (20). Upregulation Azasetron HCl of miR-181a and miR-335 has been observed in AML patients transporting gene mutations (21, 22). And, very recently, miR-3151 was reported to independently affect the outcome of patients with cytogenetically normal AML (23). In the present study, we analyzed the mechanisms of CXCR4 signalingCmediated chemoresistance and exhibited that the human miRNA let-7a, which negatively regulates BCL-XL expression, is regulated by SDF-1/CXCR4 signaling in human AML cells. In our experiments, inhibiting CXCR4 or overexpressing let-7a in AML cells led to reduced expression of BCL-XL and enhanced cytarabine-induced (Ara-CCinduced) apoptosis both in vitro and in vivo. Results let-7a in OCI-AML3 cells is usually downregulated by SDF-1 treatment and upregulated with CXCR4 antagonist. To explore how CXCR4-mediated signaling in AML cells elicits chemoresistance, we first performed a miRNA microarray platform (see Methods). OCI-AML3 cells were treated with 100 ng/ml SDF-1 or 250 nM Azasetron HCl POL6326 (a CXCR4 antagonist), total RNA was extracted at specific time points (0, 1, 2, 4, and 8 hours), and miRNA expression profiling was performed. 47 miRNA probes were identified to be significantly changed in either direction with treatment (significant at 0.01 level of the univariate test; Physique ?Physique1A).1A). We focused on miRNAs that could potentially function as tumor suppressors and connect CXCR4 signaling to chemoresistance. The let-7a coding sequence was selected for further analysis because it was not only one of the most strongly downregulated miRNAs in OCI-AML3 cells after SDF-1 treatment,.