Elevated miR-155 Expression Research Articles

Hypoxia-induced microRNA-155 overexpression in extracellular vesicles promotes renal cell carcinoma progression by targeting FOXO3.

Renal cell carcinoma (RCC) is a form of cancer arising from the renal epithelium, with high mortality rates that have reached stable levels over the past decade. The tumor microenvironment is an essential regulator of tumor progression and survival, and extracellular vesicles (EVs) are an important facet of this microenvironment. Herein, we explored the impact of hypoxia-induced miR-155 expression in EVs on FOXO3 expression in RCC cells and their associated oncogenic activity. We found that RCC patients exhibited elevated miR-155 expression in EVs relative to healthy controls, suggesting that this miRNA may be important in the context of RCC progression. We then characterized EVs produced from RCC cell lines (Caki-1 and 786-O) under normoxic and hypoxic conditions, revealing that hypoxia-induced EVs contained higher levels of miR-155 and promoted cell proliferation. Then, we identified FOXO3 as a miR-155 target. Lastly, hypoxia-induced EVs were found to be able to significantly inhibit FOXO3 activation via facilitating miR-155 up-regulation. Together, these findings indicate that hypoxia can promote the upregulation of miR-155 in EVs and that this miRNA can act in RCC cells to suppress FOXO3 expression, thereby enhancing cellular tumor progression.

Cobomarsen, an Oligonucleotide Inhibitor of miR-155, Slows DLBCL Tumor Cell Growth In Vitro and In Vivo.

miRNA-155 is an oncogenic miRNA highly expressed in B-cell malignancies, particularly in the non-germinal center B-cell or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL), where it is considered a potential diagnostic and prognostic biomarker. Thus, miR-155 inhibition represents an important therapeutic strategy for B-cell lymphomas. In this study, we tested the efficacy and pharmacodynamic activity of an oligonucleotide inhibitor of miR-155, cobomarsen, in ABC-DLBCL cell lines and in corresponding xenograft mouse models. In addition, we assessed the therapeutic efficacy and safety of cobomarsen in a patient diagnosed with aggressive ABC-DLBCL. Preclinical studies included the delivery of cobomarsen to highly miR-155-expressing ABC-DLBCL cell lines to assess any phenotypic changes, as well as intravenous injections of cobomarsen in NSG mice carrying ABC-DLBCL xenografts, to study tumor growth and pharmacodynamics of the compound over time. To begin to test its safety and therapeutic efficacy, a patient was recruited who underwent five cycles of cobomarsen treatment. Cobomarsen decreased cell proliferation and induced apoptosis in ABC-DLBCL cell lines. Intravenous administration of cobomarsen in a xenograft NSG mouse model of ABC-DLBCL reduced tumor volume, triggered apoptosis, and derepressed direct miR-155 target genes. Finally, the compound reduced and stabilized tumor growth without any toxic effects for the patient. Our findings support the potential therapeutic application of cobomarsen in ABC-DLBCL and other types of lymphoma with elevated miR-155 expression.

Association of miR-155 and Angiotensin Receptor Type 1 Polymorphisms with the Risk of Ischemic Stroke in a Chinese Population.

There is increasing evidence suggesting that dysregulation of miR-155 and its target angiotensin receptor type 1 (AT1R) are linked to the incidence of ischemic stroke (IS), but the underlying mechanisms remain to be clarified. In this study, we therefore sought to investigate how miR-155 and AT1R polymorphisms affect IS risk. We included 579 IS patients and 509 age-matched controls in the present analysis, genotyping individuals for the rs767649 polymorphism in miR-155, as well as for the rs1492099 and rs275653 polymorphisms in AT1R via iMLDR-TM genotyping technology. The allele and genotype frequencies for the assessed polymorphisms were comparable in IS patients and controls, without any detectable association between AT1R haplotype and IS risk. We conducted additional trial of ORG 10172 in acute stroke treatment-mediated stratification, which indicated that the AT1R rs1492099 T allele was linked to a decreased risk of large-artery atherosclerosis (LAA) stroke. We further found that those with the AT1R rs275653 AA genotype had a decreased risk of small-artery occlusion (SAO) strokes. We further confirmed elevated miR-155 expression in IS patients, but observed no link between the rs767649 polymorphism and expression of this microRNA. Similarly, rs1492099 and rs275653 polymorphisms did not impact AT1R expression levels. The miR-155 rs767649 polymorphism does not seem to be a key determinant of IS risk, whereas the AT1R rs1492099 polymorphism is linked to reduced LAA-stroke risk, and the rs275653 AA genotype is potentially protective against SAO strokes.

MicroRNA-155 inhibition up-regulates LEPR to inhibit osteoclast activation and bone resorption via activation of AMPK in alendronate-treated osteoporotic mice.

Osteoporosis is characterized by a progressive increase in bone fragility, leading to low bone mass and structural deterioration of bone tissue. MicroRNA-155 (miR-155) is highly expressed in osteoporosis. Thus, the current study aimed to investigate the effect of miR-155 on the inhibition of osteoclast activation and bone resorption by targeting leptin receptor (LEPR) through the adenosine monophosphate activated protein kinase (AMPK) pathway in alendronate-treated osteoporotic mice. An osteoporosis mouse model was established to examine the bone tension and bone density and the expression of miR-155 in osteoclasts. Binding sites between miR-155 and LEPR were verified. Osteoclasts in the treatment group were transfected with different mimic, inhibitor, vector, or siRNA for subsequent experiments. The expression of miR-155, LEPR, AMPK, p-AMPK, RANKL, OPG, M-CSF, RANK, TRAP, Bax, Bcl-2, and the contents of TNF-α and IL-1β were all examined. The proliferation and bone resorption of osteoclasts were also detected. Mice with osteoporosis exhibited decreased bone density and bone tension, along with elevated expression of miR-155. LEPR was verified as a target gene of miR-155. Down-regulated miR-155 was found to increase the expression of LEPR, AMPK, p-AMPK, OPG, Bax, decrease expression of TNF-α, IL-1β, RANKL, M-CSF, RANK, TRAP, Bcl-2, inhibit the cell proliferation and bone resorption of osteoclasts. Taken together, decreased miR-155 up-regulated LEPR via activation of AMPK, which ultimately repressed osteoclast activation and bone resorption of osteoclasts in alendronate-treated osteoporotic mice.

MiR-155 regulates the proliferation and apoptosis of pancreatic cancer cells through targeting SOCS3.

Reduced expression of suppressors of cytokine signaling 3 (SOCS3) is associated with a variety of tumors. The elevated miR-155 expression is associated with the onset of pancreatic cancer. Bioinformatics analysis revealed a targeted relation between miR-155 and the 3'-UTR of SOCS3. This study investigated whether miR-155 regulates SOCS3 expression and affects the biological effects of pancreatic cancer cells. QRT-PCR was used to detect the expression of miR-155 and SCOS3 mRNA in tumor tissues and paracancerous tissues of patients with pancreatic cancer. The dual luciferase reporter gene assay validated the target interaction between miR-155 and SOCS3. Pancreatic cancer cell line SW1990 cells were divided into miR-NC group and miR-155 inhibitor group followed by an analysis of the expressions of SOCS3, p-JAK2 and p-STAT3, cell apoptosis by flow cytometry, and cell proliferation by EdU staining. Compared with adjacent tissues, miR-155 expression was increased in tumor tissues of patients with pancreatic cancer, and SOCS3 expression was decreased. There was a targeted regulatory relationship between miR-155 and SOCS3 mRNA. Compared with HPDE6-C7 cells, miR-155 expression in pancreatic cancer SW1990 and Capan-1 cells was increased, and SOCS3 expression was decreased. Transfection of miR-155 inhibitor significantly increased SOCS3 expression in pancreatic cancer SW1990 cells, decreased the expression of p-JAK2 and p-STAT3, increased cell apoptosis, and decreased cell proliferation. Increased miR-155 expression and decreased SOCS3 expression are related to the pathogenesis of pancreatic cancer. miR-155 inhibits the proliferation and apoptosis of pancreatic cancer cells by inhibition of SOCS3 expression.

Prognostic and Clinicopathological Significance of MiR-155 in Hematologic Malignancies: A Systematic Review and Meta-analysis.

Background: Aberrant miR-155 expression has been reported in various types of hematologic malignancies. However, the prognostic and clinicopathological value of miR-155 remains unclear. Here, we performed this systemic review and meta-analysis to comprehensively evaluate the prognostic and clinicopathological significance of miR-155 expression in hematologic malignancies.Methods: We systematically searched the PubMed, EMBASE, ISI Web of Science, Cochrane library databases and OVID to identify eligible studies published from Jan 1, 2008 to Aug 1, 2018. The pooled hazard ratios (HRs) and odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were used to detect the prognostic and clinicopathological role of miR-155 in hematologic malignancies.Results: A total of 18 studies including 2316 patients were enrolled in the present meta-analysis, indicating significant association between elevated miR-155 expression and poor overall survival (OS) in 2114 patients (pooled HR = 1.72, 95%CI [1.50-1.97], p<0.001). Elevated miR-155 expression level was related to shorter event free survival (EFS, pooled HR = 1.55, 95%CI [0.94-2.57], P=0.002), disease free survival (DFS, pooled HR = 1.38, 95%CI [1.13-1.68], P=0.001), progress free survival (PFS, pooled HR = 1.58, 95%CI [1.06-2.35], p<0.001) and treatment free survival (TFS, pooled HR = 1.67, 95%CI [1.16-2.39], P=0.006). Additionally, overexpression of miR-155 was found to be significantly related to FLT3/ITD presence (OR=4.751, 95%CI [3.229-6.990], P<0.001), more WT1 mutation (OR=2.090, 95%CI [1.240-3.522], P=0.006) and less CEBPA mutation (OR=0.477, 95%CI [0.286-0.794], P=0.004) in 552 AML patients.Conclusion: MiR-155 expression was found to be associated with several leukemia-related phenotype and poor prognosis in hematologic malignancies. Therefore, miR-155 overexpression might be a convinced unfavorable prognostic indicator that helps the clinical decision-making process.

An NF-\u03baB-microRNA regulatory network tunes macrophage inflammatory responses

The innate inflammatory response must be tightly regulated to ensure effective immune protection. NF-κB is a key mediator of the inflammatory response, and its dysregulation has been associated with immune-related malignancies. Here, we describe a miRNA-based regulatory network that enables precise NF-κB activity in mouse macrophages. Elevated miR-155 expression potentiates NF-κB activity in miR-146a-deficient mice, leading to both an overactive acute inflammatory response and chronic inflammation. Enforced miR-155 expression overrides miR-146a-mediated repression of NF-κB activation, thus emphasizing the dominant function of miR-155 in promoting inflammation. Moreover, miR-155-deficient macrophages exhibit a suboptimal inflammatory response when exposed to low levels of inflammatory stimuli. Importantly, we demonstrate a temporal asymmetry between miR-155 and miR-146a expression during macrophage activation, which creates a combined positive and negative feedback network controlling NF-κB activity. This miRNA-based regulatory network enables a robust yet time-limited inflammatory response essential for functional immunity.

Elevated expression of miR-155 is associated with the differentiation of CD8+ T cells in patients with HIV-1.

The differentiation and response ofCD8+ T cells is vital in host defense against human immunodeficiency virus type 1 (HIV-1). MicroRNA (miR)‑155 is an important regulator of T cell differentiation. However, the profile of miR-155 in HIV‑1 infected individuals and its association with CD8+ T cell differentiation remain to be fully elucidated. The present cross‑sectional study was performed involving 63 HIV‑1‑infected patients undergoing highly active antiretroviral therapy (HAART), 31 HAART‑naïve patients and 35 healthy controls. The levels of miR‑155 in CD8+ T cells were detected using reverse transcription‑quantitative polymerase chain reaction analysis. Subsets of CD8+ T cell differentiation were detected using flow cytometry. The results revealed that the discord controllers and HAART‑naïve patients showed higher percentages of effector and effector memory cells, and lower percentages of naïve cells (P<0.05). The levels of miR‑155 in CD8+ T cells from the HIV‑1‑infected patients were higher, particularly in the discord controllers and HAART naïve patients (P<0.01). The expression levels of miR‑155 were positively correlated with the percentages of effector and effector memory CD8+ T cells, and negatively correlated with the percentages of naïve and central memory CD8+ T cells (P<0.01). Taken together, these findings suggested that the levels of miR‑155 in CD8+ T cells of patients with HIV-1 were increased and asso-ciated with CD8+ T cell differentiation.

Expression Signature of MicroRNA-155 and its Association with Response to Treatment within Different Subtypes of B-Cell Malignancies

Objectives: Emerging evidence suggests that microRNAs could serve as non-invasive biomarkers for cancer patients. However, they are mostly not fully investigated as biomarkers in the classification, progression and prognosis of different cancers. Our study was designed to explore the microRNA-155 (miR-155) expression levels in peripheral blood mononuclear cells (PBMCs) in patients with various subtypes of B-cell malignancies. Also, we aimed to correlate between miR-155 expression levels and the diverse clinico-pathologic features as well as the prognostic fate of these patients after treatment completion. Subjects and methods: Using whole blood samples from 53 patients with B-cell malignancies and 15 apparently healthy subjects, miR-155 was extracted and profiled by quantitative real-time PCR (RT-qPCR). The B-cell malignancies patients were including 22 diffuse large B-cell lymphoma (DLBCL), 15 chronic lymphocytic leukemia (CLL), 9 follicular lymphoma patients (FL) and 7 subjects with burkitt′s lymphoma (BL). The samples were withdrawn before starting chemotherapy in addition to after completing their therapeutic courses by 6 months. Subsequently, the patients were further sub-grouped into those with partial remission, complete remission, resistant disease and relapse. Results: We found that miR-155 expression levels differentiate lymphoma entities from normal subjects (p ≤ 0.001) and its expression fold changes distinguish B-cell lymphoma subtypes from each other’s (p<0.05). In addition, miR-155 was significantly correlated with age and LDH levels. The receiver operating curve (ROC) was employed to identify the diagnostic outcomes of miR-155 in discriminating B-cell malignancies entities from each other’s (AUC=0.957 for DLBCL vs. CLL+FL and AUC=1.000 for CLL vs. FL). Otherwise, when BL was versus healthy controls the AUC was equal to 0.552. As well, we demonstrated an association between elevated miR-155 expression levels and poor response to treatment with increased cases of relapse, partial remission or resistance to treatment. Conclusions: This study suggests that miR-155 has an expression profile that differs according to B-cell malignancies subtype. Besides, miR-155 expression levels may modulate the patient’s response to treatment. These results support a role for miR-155 to provide helpful diagnostic/prognostic information in B-cell malignancies and may highlight novel pathways to be targeted for therapeutics in the future.

Functionally distinct roles for different miR-155 expression levels through contrasting effects on gene expression, in acute myeloid leukaemia

Enforced expression of microRNA-155 (miR-155) in myeloid cells has been shown to have both oncogenic or tumour-suppressor functions in acute myeloid leukaemia (AML). We sought to resolve these contrasting effects of miR-155 overexpression using murine models of AML and human paediatric AML data sets. We show that the highest miR-155 expression levels inhibited proliferation in murine AML models. Over time, enforced miR-155 expression in AML in vitro and in vivo, however, favours selection of intermediate miR-155 expression levels that results in increased tumour burden in mice, without accelerating the onset of disease. Strikingly, we show that intermediate and high miR-155 expression also regulate very different subsets of miR-155 targets and have contrasting downstream effects on the transcriptional environments of AML cells, including genes involved in haematopoiesis and leukaemia. Furthermore, we show that elevated miR-155 expression detected in paediatric AML correlates with intermediate and not high miR-155 expression identified in our experimental models. These findings collectively describe a novel dose-dependent role for miR-155 in the regulation of AML, which may have important therapeutic implications.

MiR-155 as a Biomarker in B-Cell Malignancies.

MicroRNAs have the potential to be useful biomarkers in the development of individualized treatment since they are easy to detect, are relatively stable during sample handling, and are important determinants of cellular processes controlling pathogenesis, progression, and response to treatment of several types of cancers including B-cell malignancies. miR-155 is an oncomiR with a crucial role in tumor initiation and development of several B-cell malignancies. The present review elucidates the potential of miR-155 as a diagnostic, prognostic, or predictive biomarker in B-cell malignancies using a systematic search strategy to identify relevant literature. miR-155 was upregulated in several malignancies compared to nonmalignant controls and overexpression of miR-155 was further associated with poor prognosis. Elevated expression of miR-155 shows potential as a diagnostic and prognostic biomarker in diffuse large B-cell lymphoma and chronic lymphocytic leukemia. Additionally, in vitro and in vivo studies suggest miR-155 as an efficient therapeutic target, supporting its oncogenic function. The use of inhibiting anti-miR structures indicates promising potential as novel anticancer therapeutics. Reports from 53 studies prove that miR-155 has the potential to be a molecular tool in personalized medicine.

Preclinical Development of LNA Antimir-155 (MRG-106) in Acute Myeloid Leukemia

IntroductionMicroRNAs (miRs) are deregulated in AML and play a key role in leukemogenesis. MiR-155 is one of the most frequently overexpressed miRs in AML. Higher expression of miR-155 is associated with FLT3 internal tandem duplication (FLT3-ITD) and is associated with worse outcome, independent of FLT3-ITD status. Preliminary data shows that silencing of miR-155 induces strong antileukemic effects in AML cell lines. Altogether these data support a therapeutic role for miR-155 antagonism in AML. Here, we show the in vitro and in vivo activity of MRG-106, a novel LNA antimiR-155 compound that we are developing as a potential treatment for hematological malignancies.MethodsUnconjugated, LNA-modified oligonucleotide against miR-155 (MRG-106) was developed by miRagen Therapeutics, Inc. MRG-106 was evaluated in FLT3-ITD+ AML cell lines and primary FLT3-ITD+ AML samples for impact on apoptosis and cellular proliferation using Annexin V and MTS assays. Predicted and validated targets of miR-155 were measured by qPCR and Western Blotting to assess the efficacy of miR-155 silencing. The in vivo antileukemic effect of MRG-106 was evaluated in NOD/SCID gamma mice engrafted with MV4-11 AML cells that have elevated miR-155 expression. One week after leukemic cell inoculation, the mice were separated in 3 cohorts and received either MRG-106 (n=12); LNA-scramble control (n=12); or saline (n=6).ResultsInhibition of miR-155 decreased cell proliferation in MV-4-11 and MOLM-13 cells at 48hrs (Absorbance 450 nM (A450nM): 0.5 and 0.4 vs controls 2.4 and 2.5, respectively, p<0.01). These effects were associated with increased apoptosis (2-fold increase in Annexin staining) and re-expression of the validated miR-155 target WEE1 protein. MRG-106 produced a 4-6 fold increase in apoptosis in primary samples from 3 newly diagnosed AML patients with FLT3 -ITD cytogenetically normal AML (p<0.01). In addition, inhibition of miR-155 reduced the colony forming ability of the primary blasts (average # of colonies; controls=90 vs. MRG-106=55,p<0.01). In an MV4-11 xenograft model, MRG-106 significantly increased survival compared to controls (p<0.01). Biodistribution studies confirmed adequate MRG-106 distribution to bone marrow. GLP preclinical safety studies have been completed in rats and non-human primates, demonstrating an acceptable safety profile for MRG-106.ConclusionsInhibition of miR-155 in AML cell lines and primary AML samples in vitro and in vivo induces significant antileukemic effects. These studies validate miR-155 as a therapeutic target in AML and support the testing of MRG-106 in AML patients in the context of a phase 1 clinical trial. DisclosuresDickinson:miRagen Therapeutics: Employment, Equity Ownership. Jackson:miRagen Therapeutics: Employment, Equity Ownership. Rodman:miRagen Therapeutics: Employment, Equity Ownership.

Reacting to Inflammatory Signals

Specialized cellular niches in the bone marrow (BM) modulate critical functions of the hematopoietic stem and progenitor cells, such as self-renewal, cell-fate decisions and the balance between proliferation and differentiation. Constituents of the BM niche, such as osteoblasts, endothelial cells and macrophages, respond to inflammation with secretion of pro-inflammatory cytokines, and serve as effectors of the inflammatory circuitry; thus their excessive activation can potentially impact on the regulation of hematopoietic cells, and on the initiation and progression of myeloid malignancies. Our current understanding of the mechanisms for Bcr/Ab-negative myeloproliferative neoplasia (MPN) involves recognition of driver mutations (targeting i.e. Tet2, Jak2, Mpl, or Asxl) and of an inflammatory microenvironment. However, despite accumulating evidences on the role of inflammation and of the microenvironment in the maintenance and progression of myeloid malignancies, it is still unclear how the inflamed BM niche contributes to disease establishment and progression. Similarly, the causes leading to an inflammatory microenvironment in MPN are not fully understood. We use genetically-controlled animal model of BM inflammation to study the contribution of the inflammatory microenvironment to MPN. We found that loss of Notch signaling in the BM microenvironment results in an inflammatory state of the BM niche promoting myeloproliferation and marrow fibrosis. Using this model, we discovered that loss of Notch/RBPJ signaling leads to transcriptional upregulation of the pro-inflammatory microRNA miR-155, especially in BM endothelial and mesenchymal cells, resulting in miR-155-dependent inhibition of the NF-κB inhibitor κB-Ras1. Decreased levels of kB-Ras1 resulted in the heightened and persistent activation of NF-κB and in the increased production of NF-kB dependent pro-inflammatory cytokines, leading to uncontrolled myeloproliferation. Deletion of miR-155 in the stroma of RBPJ-/- mice prevented the development of the myeloproliferative disease, and patients affected by MPN exhibit elevated expression of miR155 in their BM. Therefore, we hypothesize that the Notch/miR155/NF-kB axis regulates the level of the inflammatory tonus in the BM niche, and that persistent deregulation of this pathway may contribute to the establishment and progression of MPN. In this presentation, we will discuss our findings and ongoing studies in the context of other recent insights on the crosstalk between tumor cells and the BM niche, and their implications for disease initiation, progression and therapeutic approaches. Disclosures No relevant conflicts of interest to declare.

Elevated miR-155 expression induces immunosuppression via CD39(+) regulatory T-cells in sepsis patient.

An altered microRNA profile exists in many infectious diseases, including sepsis. CD39(+) regulatory T-cells (Tregs) have a remarkable immunosuppressive effect and play an important role in the regulation of immune balance in sepsis. However, the correlation between microRNA changes and the ratio of CD39(+) Tregs in sepsis patients has not yet been reported. The altered microRNA expression profile in sepsis patients was analyzed in this study. Moreover, the correlation between microRNAs and disease severity and prognosis was investigated. Furthermore, the correlation between microRNAs and the percentage of peripheral blood CD39(+) Tregs was investigated and further verified in an animal model. Sixty sepsis patients and 30 healthy controls were included. The difference in microRNA expression was investigated by microRNA microarray and was further confirmed by real-time quantitative PCR. The correlations between microRNA changes and the Sepsis-related Organ Failure Assessment (SOFA) score, severity of sepsis, and survival were analyzed. The percentage CD39(+) Tregs in the peripheral blood of sepsis patients was measured by flow cytometry. The correlation between microRNAs and the percentage CD39(+) Tregs was analyzed and further confirmed in a mouse sepsis model. Compared to healthy controls, sepsis patients exhibited a significantly elevated microRNA-155 (miR-155) level (p < 0.05), which was positively related to a higher SOFA score (r = 0.641, p < 0.05) and a greater severity of sepsis. The area under the receiver operating characteristic curve of miR-155 used for the prediction of 28-day survival was 0.763, with a cut-off point of 2.47. Patients with a miR-155 level >2.47 had a lower 28-day survival (p < 0.05). The miR-155 level of patients was proportional to the percentage of CD39(+) Tregs (r = 0.637, p < 0.05). After transfection with miR-155 inhibitor, the ratio of CD39(+) Tregs in mice with sepsis was significantly reduced (p < 0.05). A higher level of miR-155 indicated a more severe condition and poorer prognosis in sepsis patients. The possible underlying mechanism could be that miR-155 induces an increased percentage of CD39(+) Tregs and thus immunosuppression.

MiR-155 Is Essential for Inflammation-Induced Hippocampal Neurogenic Dysfunction.

Peripheral and CNS inflammation leads to aberrations in developmental and postnatal neurogenesis, yet little is known about the mechanism linking inflammation to neurogenic abnormalities. Specific miRs regulate peripheral and CNS inflammatory responses. miR-155 is the most significantly upregulated miR in primary murine microglia stimulated with lipopolysaccharide (LPS), a proinflammatory Toll-Like Receptor 4 ligand. Here, we demonstrate that miR-155 is essential for robust IL6 gene induction in microglia under LPS stimulation in vitro. LPS-stimulated microglia enhance astrogliogenesis of cocultured neural stem cells (NSCs), whereas blockade of IL6 or genetic ablation of microglial miR-155 restores neural differentiation. miR-155 knock-out mice show reversal of LPS-induced neurogenic deficits and microglial activation in vivo. Moreover, mice with transgenic elevated expression of miR-155 in nestin-positive neural and hematopoietic stem cells, including microglia, show increased cell proliferation and ectopically localized doublecortin-positive immature neurons and radial glia-like cells in the hippocampal dentate gyrus (DG) granular cell layer. Microglia have proliferative and neurogenic effects on NSCs, which are significantly altered by microglial miR-155 overexpression. In addition, miR-155 elevation leads to increased microglial numbers and amoeboid morphology in the DG. Our study demonstrates that miR-155 is essential for inflammation-induced neurogenic deficits via microglial activation and induction of IL6 and is sufficient for disrupting normal hippocampal development.

Correlation of Increased Expression of MicroRNA-155 in Bladder Cancer and Prognosis.

To investigate the expression level and clinical significance of microRNA-155 (miR-155) in bladder cancer. We collected 102 pairs of tissue specimens from patients with primary bladder cancer and adjacent normal bladder specimens between March 2008 and May 2013. Quantitative real-time polymerase chain reaction (QRT-PCR) was performed to detect the expression levels of miR-155. We performed univariate survival analyses using the Kaplan-Meier method and assessed statistical significance between survival curves via the log-rank test. The mean (SD) level of miR-155 expression in tissues with bladder cancer was 13.78 (4.80), which was significantly higher on average than that in adjacent normal bladder tissues (6.14 [2.26], P <.001). Progression-free survival (PFS) was significantly lower for patients with bladder cancer who had a high expression level of miR-155 (5-year survival rate, 23.0%) than those with a low miR-155 expression level (5-year survival rate, 48.9%; P <.001). We found that elevated expression of miR-155 is correlated with a poor outcome for patients with bladder cancer; this suggests that miR-155 is a potential biomarker for bladder cancer prognosis.

Epigenetic Silencing of BRCA1 Is Linked to Homologous Recombination Repair Defects and Elevated Mir-155 Expression in Myeloid Neoplasms

Myeloid neoplasms are a heterogeneous group of clonal hematological disorders ranging from chronic myeloproliferative neoplasms (MPNs) to more aggressive mixed myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPNs) and secondary acute myeloid leukemias (sAMLs). With the exception of BCR-ABL positive chronic myelogenous leukemia (CMLs), these diseases are clinically challenging to diagnose. Due to the clinical heterogeneity and lack of new biologic insights, current therapies fail to eradicate the malignant clone and alter the natural history of the disease. However, these diagnoses share overlapping features in morphologies, cytogenetics and genetic alterations suggesting common alterations in underlying disease pathways.We have previously reported genomic loss of HR genes in MPN patients linked to defective RAD51 foci induction and sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition (McDevitt et al., ASH 2011). In this study, we directly examined homologous recombination (HR) in primary human samples by measuring induction of RAD51 foci after ionizing irradiation. Freshly isolated mononuclear leukocytes from patients diagnosed with MPNs, mixed MDS/MPNs and sAMLs were collected at the Johns Hopkins Hospital from April 2012 to March 2014. 11 of the 22 samples (50%) exhibited impaired RAD51 foci induction. With little evidence for biallelic genetic inactivation of HR genes in previous work, we evaluated promoter CpG island methylation of BRCA1, which we had previously found in a pilot study of myeloid neoplasm samples (McDevitt et al., ASH 2011). Using quantitative methylation-specific PCR (qMSP) with melt curve analysis, we reported BRCA1 promoter hypermethylation in 22 of 104 samples (21.2%), and observed statistically significant down-regulation of BRCA1 transcript in samples with BRCA1 CpG methylation (p<0.05). To determine the relationship between BRCA1 epigenetic silencing and HR status, we categorized BRCA1 gene expression of the samples according to the promoter methylation and HR status. Strikingly, we noted that all BRCA1 methylated samples were defective for HR and 5/12 (41.7%) of HR defective samples had down-regulated BRCA1 levels.We next validated the role of BRCA1 repression in vitro using the AML cell line OCI-AML3 using two independent shRNAs targeting BRCA1. We found that BRCA1-silenced cells have reduced induction of RAD51 foci, recapitulating our observations in primary samples with epigenetic silencing of BRCA1. Given the selective toxicity observed with HR-deficient cancers to PARP inhibition, we treated the BRCA1 knockdown cells with the PARP inhibitor ABT-888 and observed increased drug sensitivity.Finally, we examined additional consequences of BRCA1 loss in myeloid malignancies by investigating its role in repressing miR-155. Frequent up-regulation of miR-155 in leukemia is linked to poor prognosis, and its overexpression in murine models results in myeloproliferative disorders. Our results showed a statistically significant inverse correlation between BRCA1 and miR-155 expression in patient samples (Pearson r:-0.36; p-value<0.005). We also found elevated miR-155 expression in the BRCA1-silenced OCI-AML3 cells that increased during long-term BRCA1 repression.Our results demonstrate that defective HR repair occurs in a significant subset of myeloid malignancies. This provides a rationale for PARP inhibitors in this patient subpopulation who currently lack curative therapy, a strategy currently explored in two clinical studies (J0783, PI: Keith Pratz, MD; J1051, PI: Ivana Gojo, MD). Our finding showing BRCA1 promoter methylation in samples with defective HR highlights a novel mechanism underlying HR defects in myeloid malignancies, and offers a biomarker to identify potential responders to PARP inhibition. We also provide an alternative mechanism in which BRCA1 loss contributes to disease progression, via de-repressing miR-155 linked to aberrant expansion of myeloid cells. DisclosuresNo relevant conflicts of interest to declare.

Notch-Dependent Repression of miR-155 in the Bone Marrow Niche Regulates Hematopoiesis in an NF-κB-Dependent Manner

The microRNA miR-155 has been implicated in regulating inflammatory responses and tumorigenesis, but its precise role in linking inflammation and cancer has remained elusive. Here, we identify a connection between miR-155 and Notch signaling in this context. Loss of Notch signaling in the bone marrow (BM) nichealters hematopoietic homeostasis and leads to lethalmyeloproliferative-like disease. Mechanistically, Notch signaling represses miR-155 expression by promoting binding of RBPJ to the miR-155 promoter. Loss of Notch/RBPJ signaling upregulates miR-155 in BM endothelial cells, leading to miR-155-mediated targeting of the nuclear factor κB (NF-κB) inhibitor κB-Ras1, NF-κB activation, and increased proinflammatory cytokine production. Deletion of miR-155 in the stroma of RBPJ(-/-) mice prevented the development of myeloproliferative-like disease and cytokine induction. Analysis of BM from patients carrying myeloproliferative neoplasia also revealed elevated expression of miR-155. Thus, the Notch/miR-155/κB-Ras1/NF-κB axis regulates the inflammatory state of the BM niche and affects the development of myeloproliferative disorders.

MiR-155 is associated with the leukemogenic potential of the class IV granulocyte colony-stimulating factor receptor in CD34⁺ progenitor cells.

Granulocyte colony-stimulating factor (G-CSF) is a major regulator of granulopoiesis on engagement with the G-CSF receptor (G-CSFR). The truncated, alternatively spliced, class IV G-CSFR (G-CSFRIV) has been associated with defective differentiation and relapse risk in pediatric acute myeloid leukemia (AML) patients. However, the detailed biological properties of G-CSFRIV in human CD34(+) hematopoietic stem and progenitor cells (HSPCs) and the potential leukemogenic mechanism of this receptor remain poorly understood. In the present study, we observed that G-CSFRIV-overexpressing (G-CSFRIV(+)) HSPCs demonstrated an enhanced proliferative and survival capacity on G-CSF stimulation. Cell cycle analyses showed a higher frequency of G-CSFRIV(+) cells in the S and G2/M phase. Also, apoptosis rates were significantly lower in G-CSFRIV(+) HSPCs. These findings were shown to be associated with a sustained Stat5 activation and elevated miR-155 expression. In addition, G-CSF showed to further induce G-CSFRIV and miR-155 expression of peripheral blood mononuclear cells isolated from AML patients. A Stat5 pharmacological inhibitor or ribonucleic acid (RNA) interference-mediated silencing of the expression of miR-155 abrogated the aberrant proliferative capacity of the G-CSFRIV(+) HSPCs. Hence, the dysregulation of Stat5/miR-155 pathway in the G-CSFRIV(+) HSPCs supports their leukemogenic potential. Specific miRNA silencing or the inhibition of Stat5-associated pathways might contribute to preventing the risk of leukemogenesis in G-CSFRIV(+) HSPCs. This study may promote the development of a personalized effective antileukemia therapy, in particular for the patients exhibiting higher expression levels of G-CSFRIV, and further highlights the necessity of pre-screening the patients for G-CSFR isoforms expression patterns before G-CSF administration.

Regulation of miR-155 biogenesis in cystic fibrosis lung epithelial cells: Antagonistic role of two mRNA-destabilizing proteins, KSRP and TTP

Cystic fibrosis (CF) is characterized by a massive pro-inflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyper-expression of IL-8 protein through elevated expression of miR-155. We therefore investigated what factors promote the enhanced aberrant expression of miR-155 in CF. Here we report for the first time, the role of mRNA-destabilizing inflammatory RNA-binding proteins, KSRP and TTP, in the regulation of miR-155 biogenesis in CF lung epithelial cells. We find that KSRP and TTP have an antagonistic role in miR-155 biogenesis. While KSRP promotes enhanced processing of miR-155 precursors to mature miR-155, over-expression of TTP in the CF lung epithelial cells suppresses expression of miR-155. We find that TTP induces the expression of miR-1, which appears to be a regulator of miR-155 biogenesis in CF lung epithelial cells. These data provide novel insights into the mechanisms that induce hyper-inflammatory phenotype of CF, and are potential candidates for anti-inflammatory therapeutics for CF.