Downregulation Of miRs Research Articles

MiR-18a-5p attenuates HER2-positive breast cancer development by regulating PI3K/AKT pathway

ABSTRACT Objective: Human epidermal growth factor receptor 2 positive (HER2+) breast cancer (BC) is associated with poor prognosis. This study aimed to elucidate the role of miR−18a−5p in regulation of HER2+-BC progression along with its mechanism of action. Methods: The expression of miR−18a−5p and HER2 in BC cells and tissues was analyzed using quantitative real-time PCR while protein level expression of AKT Serine/Threonine Kinase 1 (AKT), phosphorylated AKT (p-AKT), Phosphatidylinositol 3-kinase (PI3K), phosphorylated-PI3K (p-PI3K), and HER2 were assessed by western blotting. Cell Counting Kit−8, wound healing, and cell adhesion assays were used for in vitro analysis along with xenograft tumor model construction for in vivo analysis. Pearson correlation analysis and dual-luciferase reporter (DLR) assays were used to ascertain the targeting association between miR−18a−5p and HER2. Results: There was a downregulation of miR−18a−5p expression in the BC tissues and cells. Functionally, overexpression of miR−18a−5p prevented BC cells from proliferation, adherence, migration, and activation of the P-PI3K/P-AKT pathway. In vivo experiment revealed that tumor growth was suppressed when miR−18a−5p was overexpressed. In BC, HER2 overexpression increased cell proliferation, cell-cell adhesion, migration, and P-PI3K/P-AKT signaling, but overexpression of miR−18a−5p reversed this effect because of the target relationship between miR−18a−5p and HER2. Conclusion: miR−18a−5p inhibits HER2+ BC progression by targeting HER2 to inhibit PI3K/AKT pathway activation. A theoretical foundation for the identification of new therapeutic targets for HER2+ BC may be provided by the miR−18a−5p – HER2 axis.

Quercetin and Isorhamnetin Reduce Benzo[a]pyrene-Induced Genotoxicity by Inducing RAD51 Expression through Downregulation of miR−34a

Benzo[a]pyrene (B[a]P) is metabolized in the liver into highly reactive mutagenic and genotoxic metabolites, which induce carcinogenesis. The mutagenic factors, including B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) and reactive oxygen species, generated during B[a]P metabolism can cause DNA damage, such as BPDE-DNA adducts, 8-oxo-dG, and double-strand breaks (DSBs). In this study, we mechanistically investigated the effects of quercetin and its major metabolite isorhamnetin on the repair of B[a]P-induced DNA DSBs. Whole-transcriptome analysis showed that quercetin and isorhamnetin each modulate the expression levels of genes involved in DNA repair, especially those in homologous recombination. RAD51 was identified as a key gene whose expression level was decreased in B[a]P-treated cells and increased by quercetin or isorhamnetin treatment. Furthermore, the number of γH2AX foci induced by B[a]P was significantly decreased by quercetin or isorhamnetin, whereas RAD51 mRNA and protein levels were increased. Additionally, among the five microRNAs (miRs) known to downregulate RAD51, miR-34a level was significantly downregulated by quercetin or isorhamnetin. The protective effect of quercetin or isorhamnetin was lower in cells transfected with a miR-34a mimic than in non-transfected cells, and the B[a]P-induced DNA DSBs remained unrepaired. Our results show that quercetin and isorhamnetin each upregulates RAD51 by downregulating miR-34a and thereby suppresses B[a]P-induced DNA damage.

Sensitization of Anti‐EGFR/HER2 Targeted Therapy Resistance in Breast Cancer Cells by MiRNA Manipulation

The aim of this study is to explore the resistance mechanisms to anti‐epidermal growth factor receptor (EGFR) and anti‐human epidermal growth factor receptor (HER2) targeted therapies in breast cancer (BC). Previous studies have implicated the microRNA (miR)‐200 family in maintenance of the epithelial phenotype and sensitivity to EGFR targeted therapies and miR‐221/222 have been associated with epithelial to mesenchymal transition (EMT), tumor progression and therapy resistance. To investigate mechanisms of EGFR/HER2 therapy resistance in breast cancer, therapy resistant variants of the therapy sensitive SKBR3 human breast cancer cell line were created by exposure to different concentrations of the EGFR/HER2 inhibitors lapatinib and gefitinib. Characterization of therapy sensitive and resistant variants of SKBR3 demonstrated that miR‐200a is downregulated while miR‐221 and miR‐222 are overexpressed in the therapy resistant cells. Therefore, the current objective is to validate the role of these miRNAs in therapy resistance. The hypothesis is that re‐expression of miR‐200a and downregulation of miR‐221/222 will help overcome therapy resistance to EGFR/HER2 targeted therapy. Therefore, we re‐expressed miR‐200a ectopically in the therapy resistant and sensitive cells using a lentiviral vector containing miR‐200a, or a control vector. MiR‐221/222 inhibition was achieved by expressing anti‐miR‐221/222 or control inhibitors. MTT assays were used to determine the IC50s for cell viability. Both SKBR3 parental and lapatinib resistant variants with ectopic expression of miR‐200a were more sensitive to lapatinib treatment as shown by a decrease in the IC50 when treated with lapatinib. Therapy sensitive and resistant cell lines also demonstrated a decrease in cell viability when miR‐221 or miR‐222 was downregulated using miRNA inhibitors. In conclusion, overexpression of miR‐200a or downregulation of miR‐ 221/222 family reduces the malignancy of SKBR3 parental cell lines and sensitizes EGFR/HER2 therapy resistant cells. These results suggest that manipulation of miR‐200a, miR‐221 and miR‐222 can be used as therapeutic strategies in combination with HER2/EGFR therapy to reduce cancer malignancy and therapy resistance.Support or Funding InformationThis work was supported by grants from NIH/NIGMS SC3GM094824 to SD, RCMI Programs G12RR03051 to UPR MSC, MBRS Program RISE (R25GM061838) to LDB and MM, and Bright Cure Program (U54) University of Puerto Rico/MD Anderson Cancer Center to PRV.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Micro RNAs are involved in activation of epicardium during zebrafish heart regeneration

Zebrafish could be an interesting translational model to understand and improve the post-infarction trial and possible regeneration in humans. The adult zebrafish is able to regenerate efficiently after resecting nearly 20% of the ventricular apex. This process requires the concert activation of the epicardium and endocardium, as well as trans-differentiation of pre-existing cardiomyocytes that together replace the lost tissue. The molecular mechanisms involved in this activation process are not completely clarified. In this work, in order to investigate if the downregulation of these miRNAs (miRs) are linked with the activation of epicardium, the expressions of miR-133a, b and miR-1 during regeneration were analysed. qPCR analyses in whole-heart, or from distinct dissected epicardial cells comparing to regenerative clot (containing cardiomyocytes, fibroblasts and endocardial cells) by a laser-micro-dissector, have indicated that already at 24 h there is a downregulation of miRs: (1) miR-133a and miR-1 in the epicardium and (2) miR-133b and miR-1 in the regenerative clot. All the miRs remain downregulated until 7 days post-surgery. With the aim to visualize the activations of heart component in combination with miRs, we developed immunohistochemistry using antibodies directed against common markers in mammals as well as zebrafish: Wilms tumour 1 (WT1), a marker of epicardium; heat-shock protein 70 (HSP70), a chaperon activated during regeneration; and the Cardiac Troponin T (cTnT), a marker of differentiated cardiomyocytes. All these markers are directly or indirectly linked to the investigated miRs. WT1 and HSP70 strongly marked the regeneration site just at 2–3 days postventricular resection. In coherence, cTnT intensively marked the regenerative portion from 7 days onwards. miRs-1 and -133 (a,b) have been strongly involved in the activation of epicardium and regenerative clot during the regeneration process in zebrafish. This study can be a useful translational model to understand the early epicardial activation in which miRs-133a and miR-1 seem to play a central role as observed in the human heart.

Abstract 475: Role of microRNA-34a in regulating oncoprotein STMN1

Abstract MicroRNAs, as negative regulators of gene expression, play a major role in cellular homeostasis. Downregulation of miRs is common in cancer which results in upregulation of multiple oncogenes in cancer. Here we show that stathmin (STMN1), which is over-expressed and oncogenic in wide variety of cancers, is regulated by microRNA-34 in prostate cancer. Earlier studies suggest that stathmin is involved in vital cellular processes such as cell proliferation, motility and metastasis. In the present study, we show elevated expression of stathmin in metastatic prostate cancer. Functionally, knockdown of stathmin in prostate cancer cells resulted in reduced cell proliferation and invasion in vitro, and tumor growth and metastasis in vivo. We also show that tumor suppressor microRNA miR-34a targets stathmin and mir-34 is regulated by transcriptional corepressor CtBP1 (C-Terminal Binding Protein 1). Further, Microarray analyses using stathmin modulated prostate cancer cell line RNA revealed multiple molecular alterations which may play a critical role in STMN1-mediated oncogenic activity. Thus, our study suggests that CtBP1-regulated miR-34a modulates stathmin expression in prostate cancer. This study therefore demonstrates functional role of CtBP1-miR-34a-Stathmin axis in prostate cancer progression. Citation Format: Balabhadrapatruni V.S.K. Chakravarthi, Rohit Mehra, Rui Wang, Darshan Shimoga Chandrashekar, Sai Akshaya Hodigere Balasubramanya, Irfan A. Asangani, Robert J. Lonigro, Arul M. Chinnaiyan, Sooryanarayana Varambally. Role of microRNA-34a in regulating oncoprotein STMN1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 475. doi:10.1158/1538-7445.AM2017-475

Ibrutinib Responsive Micro-RNAs and Upregulation of Tumor Suppressor Targets in Chronic Lymphocytic Leukemia

Background: MicroRNAs (miRNAs, miRs) are small (~22nt) noncoding RNAs that are involved in post-transcriptional regulation of specific mRNA targets. Aberrant miRNA expression has been shown to play a role in chronic lymphocytic leukemia (CLL) pathogenesis and to have prognostic implications. The lymph node (LN) has been demonstrated to be the site of CLL cell proliferation and activation. In particular, CLL cells in the LN display significantly increased BCR and NF-kB signaling in comparison to CLL cells in the peripheral blood (PB). Ibrutinib is a clinically approved BTK inhibitor that disrupts BCR and NF-kB signaling; and has been shown to be effective in relapsed or refractory CLL. We hypothesized that 1) miRNAs would be differentially expressed between activated CLL cells in the LN and CLL cells in the PB; and 2) ibrutinib would alter miRNA expression in CLL, which may affect therapeutic response by regulating the expression of miR target genes.Methods: RNA was isolated from CD19 positive selected cells from two separate cohorts of CLL patients. The first group included 17 treatment naïve patients donating paired LN and PB CD19 positive cells (10 IGHV un-mutated [59%], 4 IGHV mutated [23.5%] and 3 IGHV unknown [17.5%]); the second group contained 38 pairs of PB CD19 positive cells (22 IGHV un-mutated [58%] and 16 IGHV mutated [42%]) before and after ibrutinib treatment on day 28. A panel of 22 miRs previously reported to play a role in CLL pathogenesis, prognosis, or BCR signaling was profiled by qPCR. The expression level of a custom panel of genes encoding putative miR targets was quantified using the nanoString nCounter assay and verified by qPCR. Functional studies were performed in MEC-1 and SP-53 cell lines, which were treated separately with 1 μM ibrutinib or 1 μM fludarabine for 48 hours. Live cells were identified by trypan blue staining; miR and mRNA expression was quantified by qPCR.Results: Nineteen of 22 (86.4%) miRs were significantly increased (p < 0.05) in CLL cells from the LN in comparison to paired CLL cells from the PB, correlating with an activated B cell phenotype. Examination of mRNA transcripts of putative miR targets in 13 paired LN and PB samples showed that multiple tumor suppressor transcripts including CYLD, ARID1B and FOXP1 were significantly decreased in LN-derived CLL cells. Assay of 22 miRs in PB CLL cells from 38 patients before and after ibrutinib treatment indicated that a subset of miRs (miR-22, miR-34a, miR-146b and miR-181b) were significantly decreased (p < 0.05) in response to ibrutinib. Quantification of mRNA transcripts of putative targets of the ibrutinib responsive miRs by the nanoString nCounter assay showed that several tumor suppressor transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) were significantly increased. Functional studies in MEC-1 and SP-53 cell lines confirmed that miR-22, miR-34a, miR-146b and miR-181b were down-regulated and the tumor suppressor targets of these miRs (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, PTEN and SMAD4) were significantly increased after ibrutinib treatment. The alteration of miR and gene expression was specific to the effects of ibrutinib as similar changes were not observed post treatment with the chemotherapeutic agent fludarabine.Conclusions: miRNA expression patterns are significantly altered between CLL cells in the LN and those in the PB. An overall increase in the expression of miRs and down-regulation of mRNAs of tumor suppressor genes (ARID1B, CYLD and FOXP1) was observed in CLL cells in the LN, correlating with an activated B-cell phenotype. Ibrutinib treatment and inhibition of BCR signaling resulted in down-regulation of miR-22, miR-34a, miR-146b and miR-181b and concomitant up-regulation of multiple tumor suppressor targets including ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4 in PB CLL cells. Similar findings were also observed in ibrutinib treated MEC-1 and SP-53 cell lines. Additional studies are needed to address how much the down-regulation of miRs and consequent up-regulation of tumor suppressors in response to ibrutinib contributes to the overall disease response and whether this effect could sensitize CLL cells to treatment with cytotoxic agents. DisclosuresWiestner:Pharmacyclics: Research Funding.

A Novel Marine Drug, SZ-685C, Induces Apoptosis of MMQ Pituitary Tumor Cells by Downregulating miR-200c

We found a novel marine drug, SZ-685C, that was isolated from the secondary metabolites of a mangrove endophytic fungus (No. 1403) collected from the South China Sea, which has been reported to inhibit the proliferation of certain tumor cells. However, its anticancer mechanism remains unknown. The aims of this study were to observe the effectiveness of SZ-685C on pituitary adenoma cells and determine the underlying mechanisms of action. A rat prolactinoma cell line, MMQ, was used in this study. A dose escalation of SZ-685C was performed on this cell line, and cell viability was assessed using an MTT assay. Hoechst 33342, Annexin V-FITC/PI, TUNEL staining and flow cytometry were used to evaluate the extent of apoptosis at each concentration of SZ-685C. The effect of SZ-685C on prolactin expression was also evaluated using RT-PCR and immunoblotting. Quantitative RT-PCR was used to detect the expression of miR-200c in SZ-685C-stimulated MMQ cells and pituitary adenoma tissues. This miRNA was then overexpressed in MMQ cells via transfection of a miR-200c mimic to identify the mechanism underling the anti-tumor effect of SZ-685C. SZ-685C inhibited MMQ cell growth in a dose-dependent manner but showed little toxicity toward rat pituitary cells (RPCs). The IC50s of SZ-685C in MMQ cells and RPCs were 13.2 ± 1.3 mM and 49.1 ± 11.5 mM, respectively, which was statistically significant. Increasing numbers of apoptotic cells were observed in response to escalating concentrations of SZ-685C, and the expression level of prolactin (PRL) was inhibited. Nevertheless, the level of PRL mRNA was unchanged. Additionally, miR-200c was upregulated in MMQ cells compared with RPCs, and downregulation of miR- 200c was observed in SZ-685C-treated MMQ cells. Furthermore, the overexpression of miR-200c weakened the effect of SZ-685C-induced apoptosis of MMQ cells. Our results suggest that SZ-685C induces MMQ cell apoptosis in a miR-200c-dependent manner. Therefore, SZ-685C might be a useful alternative treatment for pituitary adenoma.

Regulation of Selected MicroRNAs in Multiple Myeloma,

Abstract 3916 Introduction:Multiple myeloma is clinically and biologically heterogeneous. Certain translocations and chromosomal losses (t{14;16}, t{14;20}, del 17p) and gene expression profiles define high-risk disease. Recently, several groups found microRNAs (miRs) dysregulated in multiple myeloma. A profile composed of 28 miRs was found to define high-risk disease. Among the dysregulated miRs, miR15a, miR16 were down-regulated, miR19b, miR20a, miR181b were increased according to most publications. MiR-21 was generally upregulated in high-risk disease and inducible by interleukin-6. We hypothesized that commonly administered treatments for multiple myeloma would alter the expression pattern of these miRs.Materials and Methods: For these in-vitro experiments, 4 established cell lines were used: RPMI8226, OPM-2 (t4;14), Kas-6 (IL-6-dependent) and MM1-S (t14;16). The cells were treated with ionizing radiation (3- 6 Gy), lenalidomide (10 μM), doxorubicin (50 ng/ml), bortezomib (2- 50 nM), SAHA (1–3x 10−6 M), pegylated interferon α (3–300 ng/ml) and nutlin-3 (10 μM) between 2 and 48 hours. RNA was extracted and quantitative real-time RT-PCR was performed for miR-15a, miR-16, miR19b, miR-20a, miR21, miR-181b and a control gene (U6). The expression was calculated and compared by the ΔΔ CT method. Results:Ionizing radiation increased miR15a in 1/2 cell lines at early time points, increased miR-19b at early time points in 2/2 cell lines (decreased later) and increased MiR20a in 2/2 cell lines at early time points. Lenalidomide induced miR15a in 2/4 cell lines, miR19b in 3/ 4 cell lines and miR-20a in 3/ 4 cell lines. Doxorubicin increased miR-16 in 2/3 cases and miR-20a in 2/3 cases (in 1 cell line decreased). Bortezomib overall induced few changes in miR-expression. SAHA induced miR-15a in 2/3 cell lines and decreased miR-16 in 1/3. MiR-19a was decreased with SAHA in 2/4 and increased in 1/4 cell lines. MiR-20 decreased in 1/4 and increased in 1/4. MiR-21 decreased in 1 and increased in 1/4 SAHA-treated cell lines. MiR-181b increased in 2/4 cell lines. Pegylated interferon decreased MiR-15a in 3/4 cell lines, decreased miR-16a in 3/4 cell lines, increased miR19b in 2/4 cell lines. MiR-20a was increased in 2/4 and decreased in 1/4 cell lines. MiR-181b was decreased in 2/ 4 cell lines. Nutlin-3 increased miR16 in 1/3 cell lines, increased miR-20a in 2/4 cell lines, increased miR-181b in 2/4, decreased miR-181b in 1/4 cell lines. Most changes observed are in the range of −50 – + 200%. Conclusions:Many miRs are induced at early time points under non-cytotoxic conditions. The variability observed in these experiments may be due to the genetic heterogeneity of the cell lines. Interferon mostly down-modulates the expression of the miRs studied. Previous experiments, for example using endothelial cells also showed an induction of certain miRs after cytotoxic or cytostatic treatments. This can be explained as a stress response or protective mechanism enhancing tumor cell survival. However, the functional relevance of our data was not investigated. The downregulation of miRs following interferon treatment is surprising and would argue for a combination of interferon with cytostatic treatments. If confirmed using CD138 selected samples from patients with multiple myeloma, our data may be used to develop a treatment profile which ultimately might prognosticate treatment response. Our results are also relevant for future miR-based treatments for multiple myeloma. Disclosures:Orlowski:Onyx Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 4951: 2-Methoxyestradiol (2-ME2) induces microRNA alterations in prostate cancer cells: Role for miRs in 2-ME2-induced biological effects

Abstract Prostate cancer is the second most common cause of death related to cancer in Western societies. 2-Methoxyestradiol (2-ME2), an endogenous metabolite of 17-β estradiol inhibits tumor cell proliferation in various cancer cells, including the prostate. Previous studies from our laboratory showed that 2-ME2 (i) inhibits proliferation of both androgen responsive and independent cells through induction of apoptosis involving G2/M check point block; (ii) prevents the development of prostate tumors in transgenic adenocarcinoma of mouse prostate (TRAMP) model. Although various molecular targets have been proposed, the mechanism of action behind its antiproliferative activity is still uncertain. Here we investigated the possible role for 2-ME2 induced antiproliferative activity by examining the altered regulation of microRNA (miRNAs). MicroRNAs are small non-coding RNAs that down regulate gene expression during cellular processes including proliferation and apoptosis. We compared the microRNA profile in non-tumorigenic (RWPE-1), androgen responsive (LNCaP) and androgen independent (PC-3) cells in response to 2-ME2 treatment in the presence and absence of androgen-stimulation (only in LNCaP cells) using the stem-loop RT-PCR based Taqman® Array Human MicroRNA Cards (Array A and B) representing 754 unique assays specific to human mature miRNAs (Applied Biosystems, Foster City, CA). Analysis of these data identified expression of only 50% of miRs in prostate cancer cells. About 26% and 12% of these are upregulated; 20.5% and 26% were down regulated in LNCaP and PC-3 cells respectively. However androgen stimulation of LNCaP cells resulted in upregulation of 34.2% and 15% down regulation of miRs whereas 2-ME2 treatment down regulated 28.7% of miRs. Target transcripts for these miRs have been identified. To the best of our knowledge this is the first study to report in-depth investigation of global miR changes in response to 2-ME2 induced biological effects. These data demonstrate that 2-ME2 suppresses proliferation and induces apoptosis in prostate cancer cells through modulation of microRNA signature. Supported by NIH CA 135451 (APK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4951. doi:10.1158/1538-7445.AM2011-4951

MicroRNA expression profiling of human metastatic cancers

1059 Small non-coding microRNAs (miRNAs) contribute to cancer development and progression and are differentially expressed in normal tissues and cancers. To test the hypothesis that a specific miRNA expression signature characterizes the metastatic phenotype of solid tumors, we performed miRNA micro-array analysis on 43 paired primary tumors and lymphnode metastasis. Specimens were collected at the Department of Pathology, Thomas Jefferson University from 43 patients consisting in 13 breast carcinomas, 10 lung cancers, 10 bladder urothelial cell carcinomas and 10 colon cancers. 5 μg of total RNA from each sample were used. Hybridization of biotin-labeled complementary DNA was performed on a two new miRNA microarray chip (OSU_CCC version 3.0 and 4.0), which contains ∼1,100 miRNA probes (326 human and 249 mouse miRNA genes, spotted in duplicates). We identified a common metastatic cancer miRNA signature mainly composed by over-expressed miRNAs. Some of these miRNAs (miR-10b, miR-20a, miR-21) have a well characterized association with cancer progression. In particular, in metastatic breast cancers there was an up-regulation of miR-450a, miR-148a and a down-regulation of miR- 205, miR-130b. In metastatic lung cancer there was an up-regulation of miR-369–3p and a down-regulation of miR-138–1. In metastatic bladder cancer there was an up-regulation of miR-29a, miR-10b, miR-126 and a down-regulation of miR-145, miR-143, and in metastatic colon cancer there was an up-regulation of miR-138, miR-21 and a down-regulation of miR-20a and miR-204. Moreover, in the metastatic samples we found that miRNAs associated to inflammation and immune response were over-expressed due to the partial contamination of our samples by the lymphocytes. The over-expression of most miRNAs that we found over-expressed in the primary tumors had been previously reported in published signatures of the corresponding normal tissues, thus confirming that miRNA signatures are a potent tool in classifying tissues of different origin.Our results indicate that specific miRNAs may be directly involved in cancer metastasis and that they may represent a novel diagnostic tool in the characterization of metastatic cancer gene targets. No significant financial relationships to disclose.

MicroRNA (miR) expression signatures in molecular subsets of cytogenetically normal (CN) acute myeloid leukemia (AML): A Cancer and Leukemia Group B (CALGB) study

7010 Background: Aberrantly expressed miRs contribute to malignant transformation by hybridizing to specific mRNA targets and suppressing their translation into proteins. In CN AML, the largest cytogenetic AML subset, several molecular markers are associated with favorable [e.g., NPM1 mutation (mutNPM1)] or unfavorable [e.g., FLT3 internal tandem duplication (ITD) or high ERG expression (↑ERG)] outcome. We explored if differentially expressed miRs are associated with the aforementioned markers. If so, this might indicate a role for miRs in leukemogenesis and prognosis in CN AML. Methods: Diagnostic samples from 100 adults &lt;60 years, treated on CALGB 19808 (Blood 2005; 106:122a), were analyzed on miR microarrays. Among 305 miR probes, we identified differentially expressed miRs (P&lt;0.005) when comparing patients (pts) with or without mutNPM1, FLT3 ITD or ↑ERG. Results: Nine miRs were differentially expressed in FLT3 ITD compared with FLT3 wild-type pts. miR 155, previously reported in aggressive cancers, was upregulated in FLT3 ITD pts. Strong signatures were associated with mutNPM1 (42 miR probes) and ↑ERG groups (54 miR probes). Consistent with their better prognosis, pts with mutNPM1 had upregulated miRs 10 and 196 targeting HOX genes, miRs 15 and 16 targeting BCL2 and let-7 targeting RAS. Consistent with a more aggressive phenotype, ↑ERG pts had downregulation of let-7 and miR 15 and 16 but also miRs 30, 103, and 24 which target, respectively, CDK6, FBXW11 and MAPK14, all involved in cell cycle regulation and proliferation. Four miRs were associated with event free survival (EFS); all were also associated with ↑ERG. High expression of miRs 124, 129, and 194 was significantly associated with worse EFS and ↑ERG. Low expression of miR 181 was associated with worse EFS and ↑ERG. Notably, downregulation of mir 181 has also been reported in pts with aggressive B-cell chronic lymphocytic leukemia. Conclusions: We report the first miR expression signatures within molecular subsets of CN AML. Further study is warranted to elucidate mechanisms by which miRs contribute to leukemogenesis and impact prognosis, and to determine whether miRs can be therapeutically targeted. No significant financial relationships to disclose.