Mir-130a-Mediated Downregulation of SMAD4 Contributes to Reduced Sensitivity to TGFβ Stimulation in Promyelocytic Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3383-3383
Author(s):  
Mattias Hager ◽  
Corinna Cavan Pedersen ◽  
Maria Torp Larsen ◽  
Mette Klarskov Andersen ◽  
Christoffer Hother ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Noncoding Rna ◽  
Conflicts Of Interest ◽  
Luciferase Reporter ◽  
Microrna Target ◽  
Smad4 Protein ◽  
Acute Myeloid

Abstract Abstract 3383 MicroRNAs (miRNA) are noncoding RNA molecules that regulate the synthesis of proteins and, if dysregulated, can result in development of various form of cancers. We have found that miR-130a is highly expressed in immature proliferating granulocytic precursors. In more mature granulocytes the miR-130a expression is significant lower. In acute myeloid leukemia the granulocyte precursors have lost the ability to undergo terminal maturation, leading to accumulation of non-functional, immature granulocytes (myeloblasts). We hypothesize that a sustained high expression of miR-130a during granulopoiesis may sustain continuous cell proliferation. TGF-β is a strong inhibitor of cell proliferation and lack of TGF-β expression is associated with various form of cancer. Smad4 is an essential compound in the TGF-β signaling pathway. Using microRNA target-prediction software, we identified Smad4 as a putative target for miR-130a. This was confirmed experimentally by demonstrating that transient overexpression of miR-130a results in reduction in the amount of Smad4 protein. Luciferase reporter constructs with the 3`-UTR of Smad4 also respond to miR-130a – an effect that is abolished by point mutations in the miRNA–binding site. In agreement, we observed that stable overexpression of miR-130a in a granulocytic cell line reduces the level of Smad4 protein, and render the cells less sensitive to TGF-β-induced growth inhibition. This was also confirmed with cell cycles analysis. Furthermore, the effect was diminished when transfecting the same clones with SMAD4 lacking the 3-‘UTR. In line with our hypothesis, the most immature granulocyte precursors demonstrate the highest expression of miR-130a is highest, and the lowest expression of Smad4 protein. As the granulocyte precursors mature, the expression of miR-130a decreases dramatically whereas the level of Smad4 protein expression increases demonstrating inverse correlation between miR-130a and Smad4 protein. The level of Smad4 mRNA is comparable at all stages of granulopoiesis. High miR-130a levels and low or no expression of Smad4 was found in primary cells from patients with acute myeloid leukemia and in a cell line (Kasumi-1) with the t(8;21)(q22;q22) chromosomal translocation. The level of Smad4 increased in Kasumi-1 cells when the endogenous level of miR-130a was inhibited by anti-miR-130a LNA. Our data indicate that miR-130a is involved in cell cycle regulation of normal and malignant granulocytic cells through engagement of Smad4 in the TGF-β-pathway. Grant acknowledgment: Lundbeck foundation, Carlsberg foundation, Swedish Research Council. Disclosures: No relevant conflicts of interest to declare.

Effect of Down-Regulation of miR-23b-3p on the Differentiation of Acute Myeloid Leukemia via Wilms Cancer Gene 1

2021 ◽  
Vol 11 (7) ◽  
pp. 1377-1382
Author(s):  
Lixia Cao ◽  
Jing Zhang ◽  
Huijuan Ren ◽  
Yanqiu Han
Keyword(s):  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
Hot Spot ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Down Regulation ◽  
Blank Group ◽  
Acute Myeloid

miRNA has always been a hot spot research. We assessed the effect of down-regulation of miR-23b-3p on the differentiation of acute myeloid leukemia (AML). Human AML cell line U937 was divided into blank group, NC group and miR-23b-3p low expression group (transfected with miR-23b-3p inhibitor) and miR-23b-3p followed by analysis of WT1 level and relationship between miR-23b-3p and WT1 by dual luciferase reporter assay. All-trans retinoic acid is used to induce differentiation, and then the morphological changes of cells and CD11b level were detected. When miR-23b-3p level was reduced, WT1 mRNA and protein level was also decreased. Dual luciferase assay showed that miR-23b-3p bound to WT1 3’-UTR. Inhibition of miR-23b-3p significantly decreased cell proliferation. Swiss Giemsa staining showed that most of cells were in the differentiation stage with low miR-23b-3p expression. The differentiation marker CD11b was significantly higher than other groups, indicating that low miR-23b-3p expression can promote cell differentiation and reduce cell proliferation to a certain extent. Under low miR-23b-3p expression, the positive rate of CD11b was significantly increased. Down-regulating miR-23b-3p can inhibit WT1 to a certain extent and promote the differentiation of AML, which provides a guidance for the gene-level treatment of AML.

scholarly journals Knockdown of Long Noncoding RNA HOXA-AS2 Suppresses Chemoresistance of Acute Myeloid Leukemia via the miR-520c-3p/S100A4 Axis

2018 ◽  
Vol 51 (2) ◽  
pp. 886-896 ◽  
Author(s):  
Xiaoya Dong ◽  
Zhigang Fang ◽  
Mingxue Yu ◽  
Ling Zhang ◽  
Ruozhi Xiao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Online Programs ◽  
Acute Myeloid

Background/Aims: Among different molecular candidates, there is growing data to support that long noncoding RNAs (lncRNAs) play a significant role in acute myeloid leukemia (AML). HOXA-AS2 is significantly overexpressed in a variety of tumors and associated with anti-cancer drug resistance, however, little is known regarding the expression and function of HOXA-AS2 in the chemoresistance of AML. In this study, we aimed to determine the role and molecular mechanism of HOXA-AS2 in adriamycin-based chemotherapy resistance in AML cells. Methods: Quantitative real-time PCR was used to detect HOXA-AS2 expression in the BM samples and ADR cell lines, U/A and T/A cells. Furthermore, the effects of HOXA-AS2 silencing on cell proliferation and apoptosis were assessed in vitro by CCK8 and flow cytometry, and on tumor growth in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in AML. Results: In this study, we showed that HOXA-AS2 is significantly upregulated in BM samples from AML patients after treatment with adriamycin-based chemotherapy and in U/A and T/A cells. Knockdown of HOXA-AS2 inhibited ADR cell proliferation in vitro and in vivo and promoted apoptosis. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3’-UTR with complementary binding sites, which was validated using luciferase reporter assay and anti-Ago2 RIP assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in ADR cells. S100A4 was predicted as a downstream target of miR-520c-3p, which was confirmed by luciferase reporter assay. Conclusion: Our results suggest that HOXA-AS2 plays an important role in the resistance of AML cells to adriamycin. Thus, HOXA-AS2 may represent a therapeutic target for overcoming resistance to adriamycin-based chemotherapy in AML.

scholarly journals Role of Circular RNA DLEU2 in Human Acute Myeloid Leukemia

2018 ◽  
Vol 38 (20) ◽  
Author(s):  
Dong-Mei Wu ◽  
Xin Wen ◽  
Xin-Rui Han ◽  
Shan Wang ◽  
Yong-Jian Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Circular Rna ◽  
Tumor Formation ◽  
Luciferase Reporter ◽  
Proliferation And Apoptosis ◽  
Human Acute Myeloid Leukemia ◽  
Acute Myeloid

ABSTRACT In the current study, we were interested in exploring the molecular mechanism of circular RNA DLEU2 (circRNA-DLEU2) (hsa_circ_0000488) and microRNA 496 (miR-496), as well as PRKACB, in human acute myeloid leukemia (AML) cell activities. The RNA expression levels of circRNA-DLEU2, hsa-miR-496, and PRKACB were assessed by quantitative real-time PCR (qRT-PCR). The proliferation and apoptosis abilities of the cells were determined by CCK8 assay and flow cytometry analysis. Target relationships between circRNA-DLEU2 and miR-496, as well as PRKACB, were analyzed by luciferase reporter assay and probe assay. Immunoblotting assays were used to detect the protein expression level of PRKACB. We also did in vivo experiments to observe tumor formation after overexpression of circRNA-DLEU2. Our data showed that circRNA-DLEU2 was upregulated in AML tissues and cells, which promoted AML cell proliferation and inhibited cell apoptosis. circRNA-DLEU2 promoted AML tumor formation in vivo. miR-496 was inhibited by circRNA-DLEU2 and was downregulated in AML tissues. circRNA-DLEU2 inhibited miR-496 expression and promoted PRKACB expression. miR-496 antagonized the effects of PRKACB on MOLM-13 cell proliferation and apoptosis. Collectively, circRNA-DLEU2 accelerated human AML by suppressing miR-496 and promoting PRKACB expression.

In Vitro Efficacy of a Novel Liposomal Formulation of a Protein Kinase C Inhibitor In the Treatment of Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3282-3282
Author(s):  
Kuan Boone Tan ◽  
Leong Uung Ling ◽  
Gigi Ngar Chee Chiu
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Drug Product ◽  
Myelogenous Leukemia ◽  
Liposome Encapsulation ◽  
Acute Myeloid

Abstract Abstract 3282 The prognosis of patients with acute myeloid leukemia (AML) remains poor, despite the use of the first-line, anthracycline- and cytarabine-based induction chemotherapy aiming to induce complete remission in patients. Given the recent findings that intensive chemotherapy may not benefit older leukemia patients who are not candidates for stem cell transplantation (Kantarjian, H. et al, Blood, 2010; DOI: 10.1182/blood-2010-03-276485) and that the monoclonal antibody-based cytotoxic agent, gemtuzumab ozogamicin, has been voluntarily withdrawn from the market, there is a pressing need to find effective treatment for recurrent AML patients who are >60 years. Safingol [(2S, 3S)-2-amino-1,3-octadecanediol] is a potential anti-cancer bioactive lipid that induces apoptosis through PKC inhibition in leukemia cells and other cancer types. Owing to its poor solubility, safingol is administered as an oil-based emulsion; however, this formulation suffers from severe hemolysis as the dose-limiting toxicity in pre-clinical models, and its toxicity profile is yet to be determined from an ongoing Phase I clinical trial for advanced solid tumors. Liposome is a commonly used drug delivery system to solubilize hydrophobic drugs. It is anticipated that liposome encapsulation of safingol would yield a viable injectable drug product without the need of toxic vehicle such as ethanol or Cremophor-EL, and would substantially reduce the hemolytic toxicity of safingol. In this study, our intention is to develop a suitable liposome formulation of safingol and to test its therapeutic efficacy using human AML cell lines and primary patient samples. Safingol could be formulated into stable liposomes using distearyolphosphatidylcholine and cholesterol with encapsulation efficiency of ∼100%. Safingol was released from the liposomes with a sustained release profile, mainly by a diffusion-controlled mechanism. The extent of hemolysis of 0.5 mM safingol could be significantly reduced from 76% to 14% through liposome encapsulation, as determined by an in vitro hemolysis assay. The cytotoxicity of liposomal safingol was tested with MTT assay on various AML cell lines representing different subtypes, including KG-1 (M1), HL-60 (M2), NB4 (M3), U937 (M5), MV4-11 (M5) and HEL (M6), as well as K562, a cell line of blast crisis of chronic myelogenous leukemia (BC-CML). All cell lines tested responded well to the treatment of liposomal safingol, with IC50 values ranging from 1.5–14 μM. Among the various AML subtypes, NB4 was found to be the most sensitive cell line with the lowest IC50 value of 1.5±0.2 μM. Importantly, liposome encapsulation of safingol did not compromise the ability of the drug to induce apoptosis as compared to the free drug, which was mediated possibly through a mechanism dependent on the generation of reactive oxygen species and caspase activation. Liposomal safingol was further tested in 10 leukemic patient samples, and the formulation was able to induce complete loss of viability of the primary cell samples at 20 μM after 72 h of treatment. Taken together, our results demonstrated the therapeutic potential of liposomal safingol for the treatment of various AML subtypes. Further evaluation of the pharmacokinetics and the efficacy of the formulation in animal models is warranted. Disclosures: No relevant conflicts of interest to declare.

RUNX1 and GATA2 Regulate the Expression of the SET Oncogene in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 879-879
Author(s):  
Raffaella Pippa ◽  
Ana Dominguez ◽  
Nerea Marcotegui ◽  
Raquel Malumbres ◽  
Elizabeth Guruceaga ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Promoter Region ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Minimal Promoter ◽  
Research Network ◽  
Luciferase Reporter ◽  
Prognostic Impact ◽  
Minimal Promoter Region ◽  
Acute Myeloid

Abstract INTRODUCTION. The protein SET (I2PP2A), a potent protein phosphatase 2A (PP2A) inhibitor, has been implicated in many cell processes such as DNA replication, chromatin remodeling and gene transcription, differentiation, migration, and cell-cycle regulation. In fact, SET has been described as an oncogene that regulates important signaling pathways. Our group reported that PP2A inhibition is a common event in AML, and that SET is overexpressed in 28% of acute myeloid leukemia (AML) cases, where it is associated with short overall survival. Moreover, the anti-leukemic effects of the FTY720 and OP449 PP2A-activating drugs in AML cells depend on interaction/sequestration of SET. However, despite the importance of SET overexpression and its prognostic impact in both hematological and solid tumors, there are few data about the mechanisms involved in its regulation. AIM. To characterize the functional promoter region of the SET gene, and to identify transcription factors (TFs) involved in its regulation. RESULTS. Luciferase reporter assays with five truncatedconstructs allowed us to determine a 163bp-region as the minimal promoter region of SET that contains consensus sites for several TFs. Chromatin immunoprecipitation (ChIP) assays confirmed the binding of RUNX1, GATA2, MYC, and SP1. RUNX1 and GATA2 are two essential TFs in hematopoiesis, and localized on the SET promoter when the acetylation state of both histone H3 and H4 and the tri-methylation on H3K4 is high, confirming that they both could act as positive regulators of SET transcription. In silico analysis in large series of adult patient samples with de novo AML recently published by The Cancer Genome Atlas Research Network showed a significant positive correlation between SET and RUNX1 and GATA2 at mRNA level. Furthermore, knockdown of RUNX1 and/or GATA2 triggered SET downregulation, whereas only a simultaneous overexpression of these two TFs caused a significant up-regulation of SET. Interestingly, RUNX1 interacts with GATA2 in both HL-60 and HEL cell lines. Moreover, we found that SP1 is also part of this transcription complex. Altogether, these results show that RUNX1 and GATA2, together with SP1, regulate the transcription of the SET gene. CONCLUSIONS We have defined the minimal promoter region of the SET gene, and have demonstrated that RUNX1 and GATA2 regulate its expression in AML. Moreover, our functional studies demonstrate that RUNX1 and GATA2 form a complex with SP1 that activates the transcription of SET in AML cells. This study opens new directions to further understand the mechanisms of SET overexpressing leukemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals Histone Deacetylase Inhibitors Induce microRNAs Targeting BTK in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1222-1222 ◽  
Author(s):  
Lara Rizzotto ◽  
Tzung-Huei Lai ◽  
Chaomei Liu ◽  
Arianna Bottoni ◽  
Clara D. Bloomfield ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Conflicts Of Interest ◽  
Prediction Algorithm ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a highly heterogeneous disease primarily affecting the elderly. Despite considerable progress in understanding the biology of AML, the prognosis of the majority of patients still remains poor due to lack of progress in improving therapy over the past 3 decades, thus creating the need to identify new pharmacological strategies. Bruton's Tyrosine Kinase (BTK) has been demonstrated to be functionally important in a variety of hematological malignancies and several groups have recently shown that BTK is highly expressed and constitutively active in AML cell lines and in a subset of AML patients. Moreover, treatment with ibrutinib, a covalent irreversible inhibitor of BTK, can reduce proliferation, blast adhesion to bone marrow stromal cells and migration of both AML cell lines and primary AML blasts. Validation of this target also occurred with shRNA experiments confirming the specificity of BTK for growth inhibition of AML. Dysregulation of microRNA (miRNA) expression contributes to the pathogenesis of numerous human malignancies including AML, where multiple miRNAs have been associated with specific cytogenetic and molecular subsets of the disease. Using a target prediction algorithm we identified several miRNAs as potential regulators of BTK (miR-147b, miR-210-3p, miR-425-5p, miR-1253, miR-4269, miR-4667-3p). We performed 3′UTR luciferase reporter assays in the AML cell line OCI-AML3 and found that relative luciferase activities were significantly decreased (20-30%) in cells transfected with miR-147b, miR-425-5p, miR-4269 and miR-4667-3p mimics. Flow cytometry and western blotting analysis confirmed that BTK protein expression was significantly down-regulated by all the predicted miRs except miR-1253. Previous data from our lab have shown that histone deacetylase (HDAC) 1 and 2 become recruited to multiple microRNA promoters, including the ones targeting BTK. To determine the consequence of HDAC inhibition on BTK signaling, we exposed primary blasts from 14 AML patients to 10nM of the pan-deacetylase inhibitor panobinostat (LBH589) for 48h. Our results showed that panobinostat induced a 2-23 fold increase in the expression miR-147b, miR-210-3p, miR-425-5p, and miR-4667-3p in most of the patients and a time-dependent depletion of total BTK protein and downstream BTK signaling (phospho- and total ERK and phospho- and total AKT) in about half of them. BTK protein at 24h directly correlated with BTK and HDAC1 mRNA expression at 24h and miR-210 induction at 24h inversely correlated with BTK mRNA levels at 24h. Moreover, 2 days of incubation with 10nM panobinostat is able to induce robust cell death in AML primary samples (9.6-92% apoptosis). We hypothesized that HDAC inhibitors would cooperate with kinase inhibitors of BTK to synergistically abolish BTK signaling and induce death in AML cells. Therefore, we treated OCI-AML3 and MV4-11 cell lines with 0.40 µM abexinostat, 1 µM ibrutinib for 2h alone followed by washout, or a combination of 0.40 µM abexinostat with 1 µM ibrutinib for 2h, and viability was measured at 48h. The combination of abexinostat and ibrutinib induced greater than additive cytotoxicity compared to either abexinostat or ibrutinib alone. Exposure to ibrutinib alone for 2h followed by washout inhibited the kinase activity of BTK (measured by the auto-phosphorylation on Y223) but did not affect total BTK protein. However, the combination of abexinostat and ibrutinib showed both loss of BTK kinase and decrease in total BTK protein, in addition to attenuation of BTK signaling, thus confirming the dual targeting of BTK via independent mechanisms. Our results provide the rationale for the clinical evaluation of ibrutininb in combination with histone deacetylase inhibitors in AML patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals miR-4792 Inhibits Acute Myeloid Leukemia Cell Proliferation and Invasion and Promotes Cell Apoptosis by Targeting Kindlin-3

2020 ◽  
Vol 28 (4) ◽  
pp. 357-369
Author(s):  
Yun Qin ◽  
Yu Wang ◽  
Dongbo Liu
Keyword(s):  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Luciferase Reporter ◽  
Inhibitory Effects ◽  
Induced Apoptosis ◽  
Proliferation And Invasion ◽  
Acute Myeloid ◽  
Myeloid Leukemia Cell

It has been reported that kindlin-3 expression is closely associated with progression of many cancers and microRNA (miRNA) processing. However, the effects and precise mechanisms of kindlin-3 in acute myeloid leukemia (AML) have not been well clarified. Our study aimed to explore the interaction between kindlin-3 and miR-4792 in AML. In our study, we found that the expression of kindlin-3 was dramatically increased in AML samples and cell lines, and the miR-4792 level was significantly downregulated. Interestingly, the low miR-4792 level was closely associated with upregulated kindlin-3 expression in AML samples. Moreover, introduction of miR-4792 dramatically suppressed proliferation and invasion and induced apoptosis of AML cells. We demonstrated that miR-4792 could directly target kindlin-3 by using both bioinformatics analysis and luciferase reporter assay. In addition, kindlin-3 silencing had similar effects with miR-4792 overexpression on AML cells. Overexpression of kindlin-3 in AML cells partially reversed the inhibitory effects of miR-4792 mimic. miR-4792 inhibited cell proliferation and invasion and induced apoptosis of AML cells by directly downregulating kindlin-3 expression, and miR-4792 targeting kindlin-3 was responsible for the regulation of the proliferation, invasion, and apoptosis of AML cells.

scholarly journals Mir-130a Is Aberrantly Overexpressed in Adult Acute Myeloid Leukemia with t(8;21) and Its Suppression Induces AML Cell Death

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5096-5096 ◽  
Author(s):  
Haiping Dai ◽  
Chao Ding ◽  
Suning Chen ◽  
Roderick A. F. MacLeod ◽  
Stefan Ehrentraut ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Luciferase Reporter ◽  
Event Free Survival ◽  
Free Survival ◽  
Mutational Status ◽  
Gene Assay ◽  
Acute Myeloid

Abstract Emerging evidence has revealed that miRNAs can function as oncogenes or tumor suppressor genes in leukemia. By profiling miRNA expression across a cohort (n=156) with de novo acute myeloid leukemia (AML), we identified miR-130a as significantly overexpressed in AML with t(8;21). Expression of miR-130a decreased significantly once patients with t(8;21) achieved complete remission, but increased sharply at the time of relapse. In patients with t(8;21) AML, KIT mutational status was associated with miR-130a expression - with higher expression associated with KIT activating mutations. Increased miR-130a expression in t(8;21) AML was associated with worse event-free survival, however no impact on overall survival was observed. Knockdown of RUNX1/RUNX1T1 (AML1/ETO) protein in the SKNO-1 cell line resulted in decrease of expression of miR-130a. Direct binding of RUNX1/RUNX1T1 fusion protein with the promoter sequence of miR-130a was detected with luciferase reporter gene assay. Following miR-130a knockdown, SKNO-1 demonstrated increased sensitivity to etoposide. Taken together, our data suggest that miR-130a is directly activated by RUNX1/RUNX1T1, and can be used to predict leukemia burden, event-free survival and chemotherapy sensitivity in AML with t(8;21). Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document