MicroRNA-582–3p negatively regulates cell proliferation and cell cycle progression in acute myeloid leukemia by targeting cyclin B2

Abstract Background MicroRNAs (miRNAs) function as post-transcriptional gene expression regulators. Some miRNAs, including the recently discovered miR-582–3p, have been implicated in leukemogenesis. This study aimed to reveal the biological function of miR-582–3p in acute myeloid leukemia (AML), which is one of the most frequently diagnosed hematological malignancies. Methods The expression of miR-582–3p was determined using quantitative real-time PCR in blood samples from leukemia patients and in cell lines. Cell proliferation and cell cycle distribution were analyzed using the CCK-8, colony formation and flow cytometry assays. The target gene of miR-582–3p was verified using a dual-luciferase reporter assay. The G2/M phase arrest-related molecule contents were measured using western blotting analysis. Results We found miR-582–3p was significantly downregulated in the blood samples from leukemia patients and in the cell lines. MiR-582–3p overexpression significantly impaired cell proliferation and induced G2/M cell cycle arrest in THP-1 cells. Furthermore, cyclin B2 (CCNB2) was confirmed as a target gene of miR-582–3p and found to be negatively regulated by miR-582–3p overexpression. More importantly, CCNB2 knockdown showed suppressive effects on cell proliferation and cell cycle progression similar to those caused by miR-582–3p overexpression. The inhibitory effects of miR-582–3p overexpression on cell proliferation and cell cycle progression were abrogated by CCNB2 transfection. Conclusion These findings indicate new functions and mechanisms for miR-582–3p in AML development. Further study could clarify if miR-582–3p and CCNB2 are potential therapeutic targets for the treatment of AML.

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miR-362-5p promotes cell proliferation and cell cycle progression by targeting GAS7 in acute myeloid leukemia

Human Cell ◽

10.1007/s13577-019-00319-4 ◽

2020 ◽

Vol 33 (2) ◽

pp. 405-415 ◽

Cited By ~ 1

Author(s):

Fuqun Wu ◽

Changxin Yin ◽

Junhua Qi ◽

Deyu Duan ◽

Xi Jiang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Acute Myeloid Leukemia ◽

Tumor Growth ◽

Myeloid Leukemia ◽

Cell Cycle Progression ◽

Ectopic Expression ◽

Cycle Progression ◽

Acute Myeloid

AbstractRecently, miR-362-5p has attracted special interest as a novel prognostic predictor in acute myeloid leukemia (AML). However, its biological function and underlying molecular mechanism in AML remain to be further defined. Herein, we found that a significant increase in miR-362-5p expression was observed in AML patients and cell lines using quantitative real-time PCR. The expression of miR-362-5p was altered in THP-1 and HL-60 cells by transfecting with miR-362-5p mimic or inhibitor. A series of experiments showed that inhibition of miR-362-5p expression significantly suppressed cell proliferation, induced G0/G1 phase arrest and attenuated tumor growth in vivo. On the contrary, ectopic expression of miR-362-5p resulted in enhanced cell proliferation, cell cycle progression and tumor growth. Moreover, growth arrest-specific 7 (GAS7) was confirmed as a direct target gene of miR-362-5p and was negatively modulated by miR-362-5p. GAS7 overexpression imitated the tumor suppressive effect of silenced miR-362-5p on THP-1 cells. Furthermore, miR-362-5p knockdown or GAS7 overexpression obviously down-regulated the expression levels of PCNA, CDK4 and cyclin D1, but up-regulated p21 expression. Collectively, our findings demonstrate that miR-362-5p exerts oncogenic effects in AML by directly targeting GAS7, which might provide a promising therapeutic target for AML.

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ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia

Molecular Cell ◽

10.1016/j.molcel.2021.05.008 ◽

2021 ◽

Author(s):

Daniela Di Marcantonio ◽

Esteban Martinez ◽

Joice S. Kanefsky ◽

Jacklyn M. Huhn ◽

Rashid Gabbasov ◽

...

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cell Cycle Progression ◽

Nucleotide Metabolism ◽

Cycle Progression ◽

Acute Myeloid

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Loss of ISWI ATPase SMARCA5 (SNF2H) in Acute Myeloid Leukemia Cells Inhibits Proliferation and Chromatid Cohesion

International Journal of Molecular Sciences ◽

10.3390/ijms21062073 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2073

Author(s):

Tomas Zikmund ◽

Helena Paszekova ◽

Juraj Kokavec ◽

Paul Kerbs ◽

Shefali Thakur ◽

...

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cell Cycle Progression ◽

Mitotic Division ◽

Cycle Progression ◽

Hematopoietic Stem ◽

Chromatid Cohesion ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.

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RNAi-Based Functional Genomics Screening of the Human Kinome Identifies Major Growth Regulating Kinases in Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v112.11.2951.2951 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2951-2951

Author(s):

Raoul Tibes ◽

Ashish Choudhary ◽

Amanda Henrichs ◽

Sadia Guled ◽

Irma Monzon ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Acute Myeloid Leukemia ◽

Gene Silencing ◽

Functional Genomics ◽

Cell Lines ◽

Myeloid Leukemia ◽

Large Scale ◽

Cell Cycle Checkpoint ◽

Acute Myeloid

Abstract In order to improve treatment strategies for Acute Myeloid Leukemia (AML), we adapted a functional genomics approach using RNAi screening to identify molecular targets that are vital to the growth of AML. Herein we report the first large-scale kinome gene silencing screen in AML. A high throughput RNAi screen was developed for the efficient siRNA transfection of AML cell lines. Eight commercially available cationic lipid-based transfection reagents were tested for their ability to transfect several AML cell lines with siRNA. These extensive transfection optimization experiments identified two AML cells lines TF-1 and ML4 with up to 95–100 and 70–75% transfection efficiency respectively. Two independent replicate kinome screens were performed on both cell lines using a siRNA library targeting 572 kinase genes with 2 siRNA/gene. At 96 hours post transfection, cell proliferation was assessed and the B-score method was used to background correct and analyze the screening data. Several siRNA to specific kinases were identified that significantly inhibit cell proliferation of up to ~40–88%. Hits were defined at two thresholds: siRNA having a B-score of <−2 providing a statistically significance of p<0.05 (confidence of > 95%) and a cutoff B-score of <−1.5 providing greater than 87% confidence for each siRNA hit. Two different kinases (2 siRNA/gene/screen) were identified as major growth regulating kinases in TF1 cells with all 4 siRNA/gene having a B-score <−2. For two additional kinases, 3/4 siRNA for each gene had a Bscore <−2. Expanding the cutoff to a B-score <−1.5 three further kinases were targeted by at least 3/4 siRNA/gene. Similar analysis using the same criteria for ML4 cells identified one kinase targeted by 3/4 siRNA at a B-score <−2, seven kinases with 2/4 siRNA <−2 and two kinases with 3/4 siRNA/gene at a B-score of <−1.5. Common hits for both cell lines with at least 6/8 siRNA per gene from 4 screens performing at a B-score <−2 identified two kinases, one of them PLK1. Applying a B-score threshold of <−1.5, we identified five kinases for which at least 5/8 siRNA/gene from 4 screens met these criteria. Kinases/genes will be presented at the meeting.Confirmation of gene silencing and validation of growth response is currently underway for a subset of genes. Among the strongest hits are siRNA targeting PLK1, as well as siRNA targeting three other kinase-genes involved in regulating cell cycle progression and checkpoints and gene ontology (GO) analysis showed enrichment in cell cycle and cell cycle-checkpoint processes. Inhibitors against PLK1 and other kinase hits identified in the screen are in (pre)-clinical development and if confirmed, our experiments provide a strong rational to test these in AML. The application of RNAi based screening is useful in the identification of genes important in AML proliferation, which could serve as targets for therapeutic intervention and guide AML drug development. Furthermore, results from these types of functional genomics approaches hold promise to be rapidly translated into clinical application.

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CREB Regulates Cell Cycle Progression through RFC3-PCNA Axis in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.881.881 ◽

2014 ◽

Vol 124 (21) ◽

pp. 881-881

Author(s):

Hee-Don Chae ◽

Bryan Mitton ◽

Kathleen Sakamoto

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukemia ◽

Promoter Region ◽

Myeloid Leukemia ◽

Cell Cycle Progression ◽

Target Genes ◽

Conflicts Of Interest ◽

Nuclear Antigen ◽

Cycle Progression ◽

Acute Myeloid

Abstract CREB (cAMP Response Element Binding protein) is a transcription factor overexpressed in normal and neoplastic myelopoiesis and regulates cell cycle progression, although its oncogenic mechanism has not been well characterized. Replication Factor C3 (RFC3), a 38 kDa subunit of the RFC complex, is required for chromatin loading of proliferating cell nuclear antigen (PCNA) which is a sliding clamp platform for recruiting numerous proteins in DNA replication and repair processes. CREB1 expression was coupled with RFC3 expression during the G1/S progression in the KG-1 acute myeloid leukemia (AML) cell line, suggesting that RFC3 and CREB1 might be target genes of E2F, a key transcriptional regulator of the G1/S progression. Though there were two potential E2F binding sites in the RFC3 promoter region, chromatin immunoprecipitation assays provided no evidence for E2F1 binding to the RFC3 promoter, whereas E2F1 could directly act on the CREB1 expression. Treatment with the cyclin-dependent kinase (CDK) inhibitor AT7519 decreased expression of CREB1 and RFC3 as well as well-known E2F target genes such as CCNE1, CCNA2 and CCNB1 in KG-1 cells. These results indicate that CREB1 overexpression, a potentially important prognostic marker in leukemia patients, may be associated with dysregulated CDK-E2F activity in leukemia. There was also a direct correlation between the expression of RFC3 and CREB1 in human AML cell lines as well as in AML cells from patients. CREB interacted directly with the CRE site in RFC3 promoter region. CREB knockdown primarily inhibited G1/S cell cycle transition, decreasing expression of RFC3 as well as PCNA loading onto chromatin. Exogenous expression of RFC3 was sufficient to rescue the impaired G1/S progression and PCNA chromatin loading [Chromatin-bound PCNA-positive cells (%), control vs. CREB-knockdown vs. CREB-knockdown with RFC3 overexpression, 8h after release from mitotic arrest: 66.87 +/– 0.90 vs. 24.77 +/– 0.99 vs. 79.17 +/– 0.12, n=3, p< 0.01, mean +/– SEM] caused by CREB knockdown. Taken together, our results suggest that RFC3 may play a role in neoplastic myelopoiesis by promoting the G1/S progression, and its expression is regulated by CREB. These data provide new insight into CREB-driven regulation of the cell cycle in AML cells, and may contribute to leukemogenesis associated with CREB overexpression. Disclosures No relevant conflicts of interest to declare.

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Paradoxical Enhancement of Leukemogenesis in Acute Myeloid Leukemia Cells with Moderately Attenuated RUNX1 Expressions

Blood ◽

10.1182/blood.v128.22.2710.2710 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2710-2710

Author(s):

Kensho Suzuki ◽

Ken Morita ◽

Shintaro Maeda ◽

Hiroki Kiyose ◽

Souichi Adachi ◽

...

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukemia ◽

Free Radicals ◽

Protein Level ◽

Myeloid Leukemia ◽

Cell Cycle Progression ◽

Leukemia Cells ◽

Cycle Progression ◽

Runx1 Expression ◽

Acute Myeloid

Abstract Although Runt-related transcription factor 1 (RUNX1), a member of RUNX transcription family, is known for its oncogenic role in the development of acute myeloid leukemia (AML), evidence from other groups support the oncosuppressive property of RUNX1 in leukemia cells, casting a question over the bidirectional function of RUNX1 and it is currently highly controversial. Here we report that the dual function of RUNX1 possibly arise from the total level of RUNX family expressions. To examine the precise mechanism of RUNX1 expression in leukemogenesis, we first prepared several tetracycline-inducible short hairpin RNAs (shRNAs) which could attenuate the expressions of RUNX1 at different levels in AML cells (MV4-11 and MOLM-13 cells). Intriguingly, while AML cells transduced with shRNAs which could down-regulate RUNX1 expression below 10% at protein level (sh_Rx1_profound) deteriorated the proliferation speed of AML cells, AML cells transduced with shRNAs which could moderately down-regulate RUNX1 expression to 70% at protein level (sh_Rx1_moderate) paradoxically promoted the cell cycle progression and doubled the growth rate of AML cells. Besides, RUNX1-moderately expressing AML patient cohort exhibited the worse outcome compared to RUNX1-high or RUNX1-low expressing cohorts (n = 187), indicating an underlying mechanism that confer growth advantage to AML cells with moderately inhibited RUNX1 expressions. To further investigate the correspondent gene in this paradoxical enhancement of oncogenesis in sh_Rx1_moderate-transduced AML cells, we performed comprehensive gene expression array and extracted genes that are highly up-regulated in RUNX1 moderate inhibition and down-regulated in AML cells transduced with sh_Rx1_profound. We hereafter focused on the top-listed gene glutathione S-transferase alpha 2 (GSTA2) and addressed the interaction of RUNX1 and GSTA2 and their functions in AML cells. Real time quantitative PCR (RT-qPCR) and immunoblotting revealed that the expression of GSTA2 was actually up-regulated in sh_Rx1_moderate-transduced AML cells and down-regulated in AML cells transduced with sh_Rx1_profound. Interestingly, equivalent level of compensatory up-regulation of RUNX2 and RUNX3 were observed in sh_Rx1_moderate- and sh_Rx1_profound-transduced AML cells, creating an absolute gap in the expression of total amount of RUNX (RUNX1 + RUNX2 + RUNX3), which was confirmed by RT-qPCR (total amount of RUNX expressions were estimated by primers amplifying the specific sequence common to all RUNX family members). Luciferase reporter assay of GSTA2 promoter and chromatin immunoprecipitation (ChIP) assay in the proximal promoter region of GSTA2 gene proved the association of RUNX family members with this genomic region. These results indicated that total amount of RUNX family expressions modulate the expression of GSTA2 in AML cells, which might results in a paradoxical outbursts of RUNX1 moderately-inhibited AML cells. Since GSTA2 catabolizes and scavenges free radicals such as hydrogen peroxide (H2O2), and decreased intracellular free radicals promote acceleration of cell cycle progression, we next measured the intracellular accumulation of H2O2 in RUNX1 inhibited AML cells. As we have expected, intracellular amount of H2O2 was decreased in sh_Rx1_moderate-transduced AML cells and increased in AML cells transduced with sh_Rx1_profound. Additive transduction of sh_RNAs targeting GSTA2 to AML cells with sh_Rx1_moderate reverted the proliferation speed to the control level, underpinning that growth advantage of moderate RUNX1 inhibition could be attributed to the GSTA2 overexpressions. Taken together, these findings indicate that moderately attenuated RUNX1 expressions paradoxically enhance leukemogenesis in AML cells through intracellular environmental change via GSTA2, which could be a novel therapeutic target in anti-leukemia strategy. Disclosures No relevant conflicts of interest to declare.

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Cell-cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia

Blood ◽

10.1182/blood-2009-08-240101 ◽

2010 ◽

Vol 115 (9) ◽

pp. 1768-1778 ◽

Cited By ~ 196

Author(s):

John A. Pulikkan ◽

Viola Dengler ◽

Philomina S. Peramangalam ◽

Abdul A. Peer Zada ◽

Carsten Müller-Tidow ◽

...

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Feedback Loop ◽

Cell Cycle Progression ◽

Cycle Progression ◽

Enhancer Binding Protein ◽

Blast Cells ◽

Acute Myeloid ◽

Transcriptional Target

Abstract Transcription factor CCAAT enhancer binding protein α (C/EBPα) is essential for granulopoiesis and its function is deregulated in leukemia. Inhibition of E2F1, the master regulator of cell-cycle progression, by C/EBPα is pivotal for granulopoiesis. Recent studies show microRNA-223 (miR-223), a transcriptional target of C/EBPα, as a critical player during granulopoiesis. In this report, we demonstrate that during granulopoiesis microRNA-223 targets E2F1. E2F1 protein was up-regulated in miR-223 null mice. We show that miR-223 blocks cell-cycle progression in myeloid cells. miR-223 is down-regulated in different subtypes of acute myeloid leukemia (AML). We further show that E2F1 binds to the miR-223 promoter in AML blast cells and inhibits miR-223 transcription, suggesting that E2F1 is a transcriptional repressor of the miR-223 gene in AML. Our study supports a molecular network involving miR-223, C/EBPα, and E2F1 as major components of the granulocyte differentiation program, which is deregulated in AML.

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Pleiotropic effects of spongean alkaloids on mechanisms of cell death, cell cycle progression and DNA damage response (DDR) of acute myeloid leukemia (AML) cells

Cancer Letters ◽

10.1016/j.canlet.2015.02.030 ◽

2015 ◽

Vol 361 (1) ◽

pp. 39-48 ◽

Cited By ~ 10

Author(s):

Fabian Stuhldreier ◽

Stefanie Kassel ◽

Lena Schumacher ◽

Sebastian Wesselborg ◽

Peter Proksch ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Death ◽

Acute Myeloid Leukemia ◽

Dna Damage Response ◽

Myeloid Leukemia ◽

Cell Cycle Progression ◽

Cycle Progression ◽

Damage Response ◽

Acute Myeloid

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MiR-335-3p inhibits cell proliferation, induces cell cycle arrest and apoptosis in acute myeloid leukemia by targeting EIF3E

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab116 ◽

2021 ◽

Author(s):

Ling Zhang ◽

Xiaozhen Wang ◽

Jieying Wu ◽

Ruozhi Xiao ◽

Jiajun Liu

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

G1 Arrest ◽

Cycle Arrest ◽

Promising Therapeutic Target ◽

Reverse Transcription Quantitative Pcr ◽

Acute Myeloid

Abstract Here, we aimed to investigate the biological roles and the regulatory mechanisms of miR-335-3p in acute myeloid leukemia (AML). We first found miR-335-3p was significantly down-regulated in blood samples from leukemia patients and cell lines using reverse transcription quantitative PCR. Through CCK-8 assay and flow cytometry, we observed that miR-335-3p overexpression significantly inhibited cell proliferation, induced cell cycle G0/G1 arrest and apoptosis in AML cell lines (THP-1 and U937). Moreover, miR-335-3p directly targets EIF3E and negatively regulated its expression. More importantly, EIF3E overexpression reversed the effects of miR-335-3p on cell proliferation, G1/S transition and apoptosis. Furthermore, miR-335-3p overexpression obviously downregulated the expression of CDK4, Cyclin D1 and Bcl-2, while upregulated the expression of p21 and Bad, which were significantly rescued by the co-transfection of pcDNA3.1-EIF3E. Collectively, our study proposes that miR-335-3p/EIF3E axis could be a promising therapeutic target to mitigate the progression of AML.

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