UTR Luciferase Reporter Assay Research Articles

MiR-122 promotes hepatic antioxidant defense of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) exposed to cadmium by directly targeting a metallothionein gene

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate target gene expression by binding to the 3'untranslated region (3'UTR) of the target mRNA. MiRNAs regulate a large variety of genes, including those involved in liver homeostasis and energy metabolism. Down-regulated levels of hepatic miR-122 were found in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) exposed to cadmium (Cd) stress. Here, we report for the first time that reduction of miR-122 post-transcriptionally increased metallothionein (MT) mRNA levels by binding to its 3'UTR, as shown by a 3' UTR luciferase reporter assay. The expression levels of miR-122 were negatively related to MT levels in GIFT under Cd stress. We performed in vivo functional analysis of miR-122 by injecting the fish with a miR-122 antagomir. Inhibition of miR-122 levels in GIFT liver caused a significant increase in MT expression, affected white blood cell and red blood cell counts, and serum alanine and aspartate aminotransferase activities, and glucose levels, all of which may help to relieve Cd stress-related liver stress. miR-122 silencing modulated oxidative stress and stimulated the activity of antioxidant enzymes. Our findings indicate that miR-122 regulated MT levels by binding to the 3'UTR of MT mRNA, and this interaction affected Cd stress induction and the resistance response in GIFT. We concluded that miR-122 plays an important role in regulating the stress response in GIFT liver. Our findings may contribute to understanding the mechanisms of miRNA-mediated gene regulation in tilapia in response to environmental stresses.

MiR-4262 promotes the proliferation of human cutaneous malignant melanoma cells through KLF6-mediated EGFR inactivation and p21 upregulation.

Alterations in the levels and functions of microRNAs (miRs) have been associated with carcinogenesis. In this study, we investigated the role and underlying mechanism of miR-4262 in the proliferation of human cutaneous malignant melanoma (CMM) cells. The expression levels of miR-4262 were significantly upregulated in cancerous tissues compared with those in matched adjacent normal tissues from 110CMM patients. miR-4262 was also regulated in five types of CMM cell lines, displaying an opposite expression pattern to that of Kruppel-like6 (KLF6), a proven tumor suppressor in several cancers other than CMM. KLF6 overexpression sharply reduced A375 cell proliferation, suppressed the activation of epidermal growth factor receptor (EGFR) and increased p21 expression levels, while knockdown of KLF6 by siRNA transfection had an opposite effect. Furthermore, KLF6 was proven to be a direct target gene of miR-4262 by bioinformatic analysis and KLF6‑3'UTR luciferase reporter assay. Finally, our data on miR-4262 mimic and inhibitor transfection indicated that miR-4262 could markedly reduce the expression of KLF6 protein and had a stimulatory effect on A375 cell proliferation. Our findings indicate that KLF6 acts as a tumor suppressor in CMM cells and miR-4262 promotes the proliferation of CMM cells through KLF6-mediated EGFR inactivation and p21 upregulation.

Differential expression of miR-184 in temporal lobe epilepsy patients with and without hippocampal sclerosis - Influence on microglial function.

Epilepsy is one of the most common neurological disorders characterized by recurrent seizures due to neuronal hyperexcitability. Here we compared miRNA expression patterns in mesial temporal lobe epilepsy with and without hippocampal sclerosis (mTLE + HS and mTLE −HS) to investigate the regulatory mechanisms differentiating both patient groups. Whole genome miRNA sequencing in surgically resected hippocampi did not reveal obvious differences in expression profiles between the two groups of patients. However, one microRNA (miR-184) was significantly dysregulated, which was confirmed by qPCR. We observed that overexpression of miR-184 inhibited cytokine release after LPS stimulation in primary microglial cells, while it did not affect the viability of murine primary neurons and primary astrocytes. Pathway analysis revealed that miR-184 is potentially involved in the regulation of inflammatory signal transduction and apoptosis. Dysregulation of some the potential miR-184 target genes was confirmed by qPCR and 3′UTR luciferase reporter assay. The reduced expression of miR-184 observed in patients with mTLE + HS together with its anti-inflammatory effects indicate that miR-184 might be involved in the modulation of inflammatory processes associated with hippocampal sclerosis which warrants further studies elucidating the role of miR-184 in the pathophysiology of mTLE.

An oyster species-specific miRNA scaffold42648_5080 modulates haemocyte migration by targeting integrin pathway

miRNAs are important gene regulators at post-transcriptional level and can modulate diverse biological processes, including immune response. Dozens of species-specific miRNAs have been identified in oyster Crassostrea gigas while their functions remain largely unknown. In the present study, an oyster species-specific miRNA scaffold42648_5080 was found responsive to LPS stimulation and might target a total of 31 oyster genes possibly involved in cell communication, cellular localization and cellular response to stimulus. Besides, in gain-of-function assay of scaffold42648_5080 in vivo, the phagocytosis (30.90% in miRNA group verse 23.20% in miRNA control group), apoptosis (3.10% in miRNA group verse 5.30% in miRNA control group) and migration rate (13.88% in miRNA group verse 21.03% in miRNA control group) of oyster haemocytes were found significantly altered after the injection of scaffold42648_5080 mimics. Among the target genes, integrin-linked kinase (CgILK) was considered crucial in cell migration and its interaction with scaffold42648_5080 was then verified both in vitro and in vivo. Consequently, a significant decrease of relative luciferase ratio was observed in CgILK 3′-UTR luciferase reporter assay after transfection of scaffold42648_5080 mimics (0.70-fold of that in blank group, p < 0.01). Meanwhile, when scaffold42648_5080 was overexpressed in vivo (5.41-fold of miRNA control group, p < 0.01), the expression of CgILK declined significantly to 0.25-fold of miRNA control group (p < 0.01). Comparatively, a significant decrease of the haemocyte migration rate (19.76% verse 34.82% in siEGFP control group, p < 0.01) was observed after knock-down of CgILK in vivo. The present study, as far as we know, for the first time revealed the immunomodulation role of an oyster species-specific miRNA, which might provide new insights into miRNA-mediated adaptation mechanism of oysters.

Dysregulation of microRNA-128 expression in WHO grades 2 glioma is associated with glioma-associated epilepsy: Down-regulation of miR-128 induces glioma-associated seizure

ObjectiveApproximately 80% of glioma patients will experience at least one seizure activity during the course of the disease, and because the etiology of glioma-related seizure is most likely multifactorial and complex, it remains poorly understood. MicroRNAs are a class of small noncoding RNAs that function as critical gene regulators. MicroRNA-128 was found to be decreased in glioblastoma, and knockout of the microRNA-128a gene could induce epilepsy in mice. Based on the Chinese Glioma Genome Atlas and previous study, we hypothesized that dysregulation of miR-128 expression may play a role in the pathogenesis of TAE in low-grade glioma. MethodsFifty-three low-grade glioma samples were analyzed for the expression levels of miR-128 using qRT-PCR, and candidate targets of miR-128 (Cacnge2, GRIK3, and GRIN2D) were detected by the 3′-UTR luciferase reporter assay. Four other miRs (miR-9, miR-192a, miR-92a, and miR-451) that showed dysregulation of glioblastoma in the CGGA data were also analyzed. ResultsThe microRNA-128 expression levels were down-regulated in low-grade glioma tissue (t-test; p=0.009). Dysregulation of miR-128 expression in low-grade glioma is associated with glioma-associated epilepsy (p=0.006). No statistical significance of miR-9, miR-192a, miR-92a, and miR-451 was found to be associated with LGG. ConclusionOur results here, together with other recent lines of evidence, indicate that miR-128 is an extremely attractive target for therapy in glioma patients with seizure.

Abstract 1924: miR-142-3p is a candidate mediator of chloride intracellular channel 4 (CLIC4) loss during squamous cancer progression

Abstract Chloride intracellular channel 4 (CLIC4) is a broadly expressed protein that has been implicated in multiple cellular processes, including cellular differentiation and apoptosis. As a soluble factor, CLIC4 translocates to the nucleus following exposure to a variety of stimuli, including TGF-β, TNF-α, and etoposide. Nuclear CLIC4 potentiates TGF-β signaling by preventing the dephosphorylation of phospho-SMAD2 and 3 and promotes growth arrest. Although its primary function is as a tumor suppressor, CLIC4 expression is progressively diminished during cancer progression and CLIC4 protein is excluded from the nucleus of many human epithelial neoplasms and squamous cell carcinoma (SCC) cell lines. To date, no gene deletions or mutations have been identified to account for this phenomenon; thus, identifying the mechanism of CLIC4 loss is our primary focus. To determine if epigenetic gene silencing is responsible, we evaluated the methylation status of the CLIC4 promoter in a progression panel (normal, papilloma, SCC) of epithelial cell lines by performing bisulfite sequencing. No differential promoter methylation was found between the cell lines, suggesting that the regulation may be post-transcriptional or post-translational. Because microRNAs (miRNAs) are known to regulate gene expression by mediating the degradation or translational repression of target mRNAs, we performed a bioinformatic analysis of the CLIC4 3’ UTR, which revealed a large number of putative targeting miRNAs. One such miRNA, miR-142-3p, has been predicted to target CLIC4 by multiple algorithms and has been shown to interact with the CLIC4 3’ UTR by high-throughput sequencing and crosslinking immunoprecipitation. We confirmed the functional activity of miR-142-3p against the CLIC4 3’ UTR by using a 3’ UTR luciferase reporter assay and a miR-142-3p mimic. miR-142-3p is high in the tumor tissue and plasma of patients with head and neck SCC and is correlated with worse prognosis, where CLIC4 expression is known to be low. Thus, miR-142-3p may be responsible for mediating CLIC4 loss in squamous lesions and warrants further investigation. Citation Format: Brandi L. Carofino, Stuart H. Yuspa. miR-142-3p is a candidate mediator of chloride intracellular channel 4 (CLIC4) loss during squamous cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1924.

An invertebrate-specific and immune-responsive microRNA augments oyster haemocyte phagocytosis by targeting CgIκB2.

Nuclear factor (NF)-κB pathway is an evolutionally conserved pathway in activating immune response, in which IκBs can repress the activation. In the present study, cgi-miR-2d, an invertebrate-specific microRNA, was proved to regulate CgIκB2 expression and haemocyte phagocytosis during bacterial infection in oyster Crassostrea gigas. The expression of cgi-miR-2d was significantly up-regulated after Vibrio splendidus challenge, while CgIκB2 transcripts decreased. Significant decreases in both luminescence and CgIκB2 3′UTR level was observed after transfection of cgi-miR-2d in CgIκB2 3′UTR luciferase reporter assay. CgIκB2 mRNA level decreased significantly (0.51-fold of control group, p < 0.05) in gain-of-function assay of cgi-miR-2d in vivo while it increased markedly (1.27-fold, p < 0.05) when cgi-miR-2d was repressed (0.10-fold, p < 0.01). A significant increase of haemocyte phagocytosis rate was observed in cgi-miR-2d overexpression group (p < 0.01), consistent with results in CgIκB2 knock-down group (p < 0.01). Moreover, the apoptosis rate of haemocytes was found significantly declined (28.57%, p < 0.01) in gain-of-function assay of cgi-miR-2d. Together, those results not only depicted the functional conservation of miR-2d family in anti-apoptosis of oysters but also highlighted its interaction with phagocytosis by modulating NF-κB pathway, which might dedicate critically to the well-balance of host immune response.

MiR156a Mimic Represses the Epithelial–Mesenchymal Transition of Human Nasopharyngeal Cancer Cells by Targeting Junctional Adhesion Molecule A

MicroRNAs (miRNAs) have been documented as having an important role in the development of cancer. Broccoli is very popular in large groups of the population and has anticancer properties. Junctional adhesion molecule A (JAMA) is preferentially concentrated at tight junctions and influences cell morphology and migration. Epithelial–mesenchymal transition (EMT) is a developmental program associated with cancer progression and metastasis. In this study we aimed to investigate the role of miRNAs from broccoli in human nasopharyngeal cancer (NPC). We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology. Among these, miR156a was expressed the most. In addition, synthetic miR156a mimic inhibited the EMT of NPC cells in vitro. Furthermore, it was confirmed that JAMA was the target of miR156a mimic as validated by 3’ UTR luciferase reporter assays and western blotting. Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic. In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3’ UTR of JAMA. These miRNA profiles of broccoli provide a fundamental basis for further research. Moreover, the discovery of miR156a may have clinical implications for the treatment of patients with NPC.

MiR-1908 Overexpression Inhibits Proliferation, Changing Akt Activity and p53 Expression in Hypoxic NSCLC Cells.

The ribosomal protein (RP)–p53 pathway has been shown to play a key role in apoptosis and senescence of cancer cells. miR-1908 is a newly found miRNA that was reported to have prognostic potential in melanoma. However, its role and mechanism in the progression of non-small cell lung cancer (NSCLC) are largely unknown. In this study, we found that expression of miR-1908 was significantly downregulated in human NSCLC cell lines, including SK-MES-1, A549, and NCI-H460. Then the role of miR-1908 in NSCLC cell proliferation was explored. The miR-1908 mimic was transfected into NSCLC cell lines, and their proliferation was detected. MTT and Cell Titer-Blue H analyses showed that the cell proliferation was notably reduced by the miR-1908 mimic transfection. Moreover, we found the RP–p53 pathway was activated by miR-1908 mimic. Moreover, the miR-1908 inhibitor transfection had a completely opposite effect on the NSCLC cell proliferation than that of miR-1908 mimic. To explore the underlying mechanism of that, TargetScan bioinformatics server and 3′-UTR luciferase reporter assay were applied to identify the targets of miR-1908. Our results showed that AKT1 substrate 1 (AKT1S1), a newly proven suppressor of the RP–p53 pathway, was a target of miR-1908, suggesting a probable mechanism for miR-191 suppressing NSCLC cell proliferation. Our findings provide a novel molecular target for the regulation of NSCLC cell proliferation.

MicroRNA-124 negatively regulates LPS-induced TNF-α production in mouse macrophages by decreasing protein stability.

MicroRNAs play pivotal roles in regulation of both innate and adaptive immune responses. In the present study, we investigated the effects of microRNA-124 (miR-124) on production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-treated mouse macrophages. Mouse macrophage cell line RAW264.7 was stimulated with LPS (100 ng/mL). The levels of miR-124 and TNF-α mRNA were evaluated using q-PCR. ELISA and Western blotting were used to detect TNF-α protein level in cell supernatants and cells, respectively. 3'-UTR luciferase reporter assays were used to analyze the targets of miR-124. For in vivo experiments, mice were injected with LPS (30 mg/kg, ip). LPS stimulation significantly increased the mRNA level of miR-124 in RAW264.7 macrophages in vitro and mice in vivo. In RAW264.7 macrophages, knockdown of miR-124 with miR-124 inhibitor dose-dependently increased LPS-stimulated production of TNF-α protein and prolonged the half-life of TNF-α protein, but did not change TNF-α mRNA levels, whereas overexpression of miR-124 with miR-124 mimic produced the opposite effects. Furthermore, miR-124 was found to directly target two components of deubiquitinating enzymes: ubiquitin-specific proteases (USP) 2 and 14. Knockdown of USP2 or USP14 accelerated protein degradation of TNF-α, and abolished the effect of miR-124 on TNF-α protein stability. miR-124, targeting USP2 and USP14, negatively regulates LPS-induced TNF-α production in mouse macrophages, suggesting miR-124 as a new therapeutic target in inflammation-related diseases.

Abstract A27: Zeb1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis

Abstract Introduction: Lung cancer is the leading cause of cancer-related death, primarily due to distant metastatic disease. Metastatic cancer cells undergo an epithelial-to-mesenchymal transition (EMT) regulated by a double-negative feedback loop between the microRNA-200 (miR-200) family and Zeb1, but the precise mechanisms of Zeb1-dependent EMT in promoting malignancy remain largely undefined. While the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. This study investigates the collaborative effect of EMT and ECM in the metastatic process. Methods: Bioinformatic analysis of TCGA dataset was done to correlate ECM-associated gene expression with EMT gene signature scores. Western blotting and qPCR analysis of epithelial and mesenchymal lung cancer cell lines were performed to determine expression levels of collagen, LOX, and LOXL2. Lung tumor tissues from non-metastatic KrasG12D and metastatic KrasG12D;p53R172H mutant mice were analyzed by immunohistochemistry, Masson's trichrome staining, and second harmonics generation for collagen, LOX, and LOXL2 expression as well as collagen fiber organization. Syngeneic primary tumors generated by subcutaneous injection of murine lung cancer cell lines were analyzed in a similar fashion. Promoter and 3′-UTR luciferase reporter assays were performed to determine direct regulation LOXL2 and LOX by Zeb1 and miR-200, respectively. Results: Our results reveal increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in Zeb1-activated mesenchymal lung cancer cells. Additionally, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and Zeb1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principle isoform driving lung cancer metastasis by crosslinking and stabilizing insoluble collagen deposition in primary tumor tissues. Conclusions: Our study demonstrates that mesenchymal lung cancer cells metastasize by modulating the compositional and structural properties of the ECM through LOXL-mediated collagen crosslinking and deposition. We are the first to validate direct regulation of LOX and LOXL2 by the miR-200/Zeb1 axis, delineate collagen as a prognostic marker for lung cancer, and identify LOXL2 as a potential therapeutic target against tumor progression. Citation Format: David H. Peng, Pan Tong, Lauren A. Byers, Jing Wang, Chad J. Creighton, Don L. Gibbons. Zeb1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A27.

Mesenchymal Stem Cells Alleviate LPS-Induced Acute Lung Injury in Mice by MiR-142a-5p-Controlled Pulmonary Endothelial Cell Autophagy

Background/Aims: Damages of pulmonary endothelial cells (PECs) represent a critical pathological process during acute lung injury (ALI), and precede pulmonary epithelial cell injury, and long-term lung dysfunction. Transplantation of mesenchymal stem cells (MSCs) has proven therapeutic effects on ALI, whereas the underlying mechanisms remain ill-defined. Method: We transplanted MSCs in mice and then induced ALI using Lipopolysaccharides (LPS). We analyzed the changes in permeability index and lung histology. Mouse PECs were isolated by flow cytometry based on CD31 expression and then analyzed for autophagy-associated factors LC3 and Beclin-1 by Western blot. Beclin-1 mRNA was determined by RT-qPCR. In vitro, we performed bioinformatics analyses to identify the MSCs-regulated miRNAs that target Beclin-1, and confirmed that the binding was functional by 3'-UTR luciferase reporter assay. Results: We found that MSCs transplantation significantly reduced the severity of LPS-induced ALI in mice. MSCs increased autophagy of PECs to promote PEC survival. MSCs increased Beclin-1 protein but not mRNA. MiR-142a-5p was found to target the 3'-UTR of Beclin-1 mRNA to inhibit its protein translation in PECs. MSCs reduced the levels of miR-142a-5p in PECs from LPS-treated mice. Conclusion: MSCs may alleviate LPS-ALI through downregulation of miR-142a-5p, which allows PECs to increase Beclin-1-mediated cell autophagy.

MiR-155 Amplifies AKT and NFkB Signaling By Targeting Multiple Regulators of BCR Signal in DLBCL

IntroductionThe B-cell receptor (BCR)- activated PI3K pathway plays a critical pro-survival function in normal B-lymphocytes and certain B-cell malignancies. Multiple mechanisms are involved in triggering and modulation of the BCR signal amplitude. MiR-155 has emerged as a positive regulator of PI3K signaling in multiple malignancies, including DLBCL, through targeting negative modulator of this pathway, SHIP-1. In the present study we have searched for new targets of miR-155, which might play a role in the deregulation of AKT and NFkB signaling in DLBCL.MethodsMiR-155 target prediction was performed with PicTar, Miranda and TagetScan algorithms. Predicted miR-155 targets were validated with 3'UTR luciferase reporter assays in HEK293 cells. MiR-155 expression was modulated through transfection with miR-155 mimic or miR-155 inhibitor. The consequences of the miR-155 perturbations were assessed in DLBCL cell lines by proliferation assays (MTS) and immunoblotting with antibodies against predicted miR-155 targets and p-AKT. The DEPTOR silencing in DLBCL cells was achieved with retroviral shRNA vector and its consequences were assessed using proliferation assays and immunoblotting. DEPTOR mRNA expression and survival of DLBCL patients was determined using publicly available microarray data (Lenz et al, 2008, GEO accession GSE10846).ResultsUsing miRNA target finding algorithms, we identified miR-155-matching sequences in 3'UTRs of two genes involved in SYK/PI3K/AKT pathway regulation: c-CBL (SYK ubiquitin E3 ligase) and DEPTOR (an mTOR phosphatase). MiR-155 suppressed the luciferase activities of vectors containing 3'UTR fragments from SHIP-1, c-CBL and DEPTOR genes with wild-type, but not mutant miR-155 seed sequence. To establish a link between miR-155 and c-CBL or DEPTOR, DLBCL cell lines were transfected with a control non-targeting miR or miR-155 mimic. The control miR did not affect the protein level of SHIP-1, c-CBL or DEPTOR, whereas introduction of miR-155 resulted in a decrease in expression of these proteins. The repression of miR-155 target genes was accompanied by increased phosphorylation of AKT. As expected, neutralization of endogenous miR-155 in U2932 cell line by anti-miR-155 exhibited opposite effects. In addition to modulation of SYK/PI3K/AKT pathway, miR-155 has been reported to modulate NFĸB activity. To determine whether miR-155 affects NFĸB in DLBCL, we assessed transcript abundance of the genes regulated by NFĸB (CD40, BFL-1, RelB, IĸBα, A20, MIR155HG) in U2932 cell line with reduced miR-155 level. Repression of endogenous miR-155 in these cells led to marked downregulation of NFĸB-controlled genes, indicating that miR-155 amplifies NFĸB signaling in this DLBCL cell line. Consistent with this, anti-miR-155 sensitized U2932 cells to ibrutinib.Since the function of a newly identified miR-155 target, the mTOR phosphatase DEPTOR, in DLBCL has not been elucidated, we silenced the expression of this protein with shRNA. Attenuated protein level of DEPTOR enhanced activity of AKT and promoted proliferation of SU-DHL4 cell line. To determine whether expression level of DEPTOR correlates with patient survival, we analyzed data from gene expression studies. Higher DEPTOR mRNA level in primary DLBCL biopsies was associated with longer overall survival (log rank test, p=0.018). Collectively, these data suggest that DEPTOR plays a tumor suppressor function in DLBCL.ConclusionsOur data underscore the role of miR-155 in the regulation of AKT and NFkB prosurvival signaling in DLBCL. MiR-155 regulates AKT signaling not only by decreasing expression of SHIP-1, but also by modulating the abundance of c-CBL and DEPTOR. DEPTOR modulates AKT activity and its silencing promotes proliferation of DLBCL cells, suggesting that DEPTOR functions as a tumor suppressor in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Histone Deacetylase Inhibitors Induce microRNAs Targeting BTK in Acute Myeloid Leukemia

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. DisclosuresNo relevant conflicts of interest to declare.

MiR-27 impairs the adipogenic lineage commitment via targeting lysyl oxidase.

The recruitment and commitment of mesenchymal stem cells and their terminal differentiation into adipocytes are the main pathways for increasing adipocyte cell numbers during obesity. Our previous studies have shown that lysyl oxidase (Lox) is upregulated and functions as an essential factor during bone morphogenetic protein 4 (BMP4) -induced C3H10T1/2 cell adipocytic lineage commitment. However, the mechanism of Lox regulation during adipogenic lineage commitment has remained largely unestablished. Samples of adipose tissue from humans with different BMI and C57BL/6 mice with a high-fat diet were used to compare microRNA-27 (miR-27) expression level associated with obesity. Taqman assays were used for miR-27 expression detection and Oil Red O staining for adipogenesis analysis. A negative correlation was identified between Lox expression level and miR-27 expression in both BMP4-treated C3H10T1/2 cells and human subcutaneous adipose tissues. A Lox 3' UTR luciferase reporter assay showed that miR-27 directly targeted Lox. Furthermore, overexpression of miR-27 impaired BMP4-induced upregulation of Lox and adipocytic commitment, which could be rescued by overexpression of mature Lox. Conversely, miR-27 inhibition by specific inhibitors increased Lox expression and adipocytic commitment. Taken together, these results suggest a novel role for miR-27 in repressing adipogenic lineage commitment by targeting Lox.

Mmu-miR-351 attenuates the survival of cardiac arterial endothelial cells through targeting STAT3 in the atherosclerotic mice

The signal transducer and activator of transcription 3 (STAT3) signaling pathway was involved in regulation of endothelial cell survival/apoptosis and was regarded as a target for prevention of atherosclerosis or other cardiovascular diseases. Factors, regulating STAT3 expression and activity, have aroused a wide range of interest, such as miRNAs or transcription factors. The aim of this study is to explore the role of miR-351, a miRNA found not long before, in the regulation of STAT3 expression and endothelial cell survival in the model mice with atherosclerosis (AS). Expression of miR-351 in the serum and cardiac arterial endothelial cells of the WT mice and AS mice was detected. Real-time qPCR analysis showed that miR-351 was upregulated in the serum and endothelial cells of the AS mice, displaying an opposite expression pattern with STAT3. To explore the role and mechanism of miR-351 in the endothelial cell survival, the miR-351 mimic was transfected in to the endothelial cells. MTT and Trypan Blue assays showed miR-351 attenuated the survival of endothelial cells. Our results of the TargetScan output and the 3′UTR luciferase reporter assay indicated that STAT3 was target of miR-351. Additionally, miR-351 resisted the elevation of STAT3 protein level and promotion of endothelial cell survival caused by SD19. Finally, our in vitro angiogenesis assay revealed that miR-351 suppressed angiogenesis and resisted the promotion of angiogenesis caused by SD19. In conclusion, miR-351 was upregulated in the atherosclerotic mice. MiR-351 can attenuate the survival of endothelial cells and suppress angiogenesis through targeting STAT3 in vitro.

ANGIOPOIETIN-1 PROMOTES ANGIOGENESIS BY MODULATING MIR-640 EXPRESION IN ENDOTHELIAL CELLS

Aberrant angiogenesis leads to numerous disorders. Angiogenic factors, such as Angiopoietin-1 (Ang-1) are important for angiogenesis. Binding of Ang-1 to its receptor Tie-2 activates mitogen-activated protein kinase (MAPK), phosphinositide 3-kinase (PI3K), and other pathways, which promote EC cell differentiation, proliferation, migration and survival. Ang-1 therapeutic potential has been proved in pre-clinical studies. However its broad spectrum of biological effects limits its use and fine tuning intracellular signals to promote the desired therapeutic actions would be a better approach. Recent studies have revealed important roles for miRNAs in regulating angiogenesis. They can be manipulated in the settings of pathological vascularization and represent a new therapeutic approach. In this study we identified miR-640 as an anti-angiogenic microRNA modulated by Ang-1. To identify new miRNA involved in Ang-1/Tie-2 signaling, HUVECs were stimulated with Ang-1 for 12, 24 and 48hrs and analysed by Illumina miRNA microarray. Array results were confirmed by RT-PCR. The biological function of miR-640 and its target ZFP91 were evaluated by monitoring tube formation by matrigel assay, apoptosis by cell count and cleaved caspase 3, and cell migration by scratch wound assay upon overexpression of the mimic, its inhibitor or siRNA against ZFP91. Target identification was done by 3’UTR luciferase assay, western blot and real-time PCR. Ang-1 treatment lead to dynamic modulation of pre-miR-640 and its mature form. Over expression of miR-640 mimic leads to down regulation of ZFP91 while inhibition of miR-640 leads to increase expression of ZFP91. As expected miR-640 mimic caused a significant loss in luciferase activity while inhibition of miR-640 restored luciferase activity when 3’UTR Luciferase Reporter assay was performed. Mutation of the seed sequence for miR-640 in ZFP91 3’UTR prevented miR-640 mimic reduction of luciferase activity Overexpression of miR-640 mimics leads to an abrogation of Ang-1 induced migration and tubulogenesis but not survival. Similarly transfecting HUVECs with siRNA against ZFP91 blunted Ang-1 induced migration and tubulogenesis. Interestingly, inhibition of endogenous miR-640 leads to a basal increase in migration and tubulogenesis. miR-640 is a new anti-angiogenic microRNA inhibiting Ang-1 induced migration and differentiation. miR-640 exerts its anti-angiogenic properties in part by modulating its target ZFP91. ZFP91 is required for Ang-1 induced migration and differentiation and appears to be a direct target of miR-640. In this study we have extended our current knowledge of pathways regulated by Ang-1 in endothelial cells and identified signaling components regulating specific biological effects.

Integrated stress response is critical for gemcitabine resistance in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with marked chemoresistance and a 5-year survival rate of 7%. The integrated stress response (ISR) is a cytoprotective pathway initiated in response to exposure to various environmental stimuli. We used pancreatic cancer cells (PCCs) that are highly resistant to gemcitabine (Gem) and an orthotopic mouse model to investigate the role of the ISR in Gem chemoresistance. Gem induced eIF2 phosphorylation and downstream transcription factors ATF4 and CHOP in PCCs, and these effects occurred in an eIF2α-S51 phosphorylation-dependent manner as determined using PANC-1 cells, and wild type and S51 mutant mouse embryo fibroblasts. Blocking the ISR pathway in PCCs with the ISR inhibitor ISRIB or siRNA-mediated depletion of ATF4 resulted in enhanced Gem-mediated apoptosis. Polyribosomal profiling revealed that Gem caused repression of global translation and this effect was reversed by ISRIB or by expressing GADD34 to facilitate eIF2 dephosphorylation. Moreover, Gem promoted preferential mRNA translation as determined in a TK-ATF4 5′UTR-Luciferase reporter assay, and this effect was also reversed by ISRIB. RNA-seq analysis revealed that Gem upregulated eIF2 and Nrf2 pathways, and that ISRIB significantly inhibited these pathways. Gem also induced the expression of the antiapoptotic factors Nupr1, BEX2, and Bcl2a1, whereas ISRIB reduced their expression. In an orthotopic tumor model using PANC-1 cells, ISRIB facilitated Gem-mediated increases in PARP cleavage, which occurred in conjunction with decreased tumor size. These findings indicate that Gem chemoresistance is enhanced by activating multiple ISR-dependent pathways, including eIF2, Nrf2, Nupr1, BEX2, and Bcl2A1. It is suggested that targeting the ISR pathway may be an efficient mechanism for enhancing therapeutic responsiveness to Gem in PDAC.

Abstract 3108: MicroRNA-200B inhibits arsenic-transformed cell migration by targeting protein kinase Cα signaling pathway

Abstract Arsenic is one of the most common environmental pollutants and long term exposure to arsenic is associated with increased risk of multiple types of cancer including lung cancer. Despite extensive studies, the mechanism of arsenic carcinogenicity has not been elucidated. MicroRNAs (miRNAs) are a large class of small non-coding RNAs and have been shown to play important roles in many fundamental processes of cancer. MiRNA-200b (miR-200b) is a member of miR-200 family, which contains 5 members. The miR-200 family has been shown to inhibit tumor cell migration; however, the underlying mechanisms are not clearly defined. We recently found that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells, which are highly migratory and invasive. To further study the mechanism by which miR-200 inhibits tumor cell migration, we investigated the effects of stably expressing miR-200b on arsenic-transformed cell migration. We found that stably expressing miR-200b drastically inhibits arsenic-transformed cell migration. Wnt5b and protein kinase Cα (PKCα) are highly expressed in arsenic-transformed cells. Stably expressing miR-200b significantly down-regulated protein levels of both Wnt5b and PKCα. Combining bioinformatics screening and 3′UTR luciferase reporter assays, we identified PKCα but not Wnt5b as a new direct target of miR-200b. We also found that siRNA knocking down Wnt5b greatly reduces PKCα activation while inactivating PKCα drastically decreases Wnt5b level, revealing a positive feedback loop between Wnt5b and PKCα. Knocking down Wnt5b or PKCα phenocopy the effect of stably expressing miR-200b on cell migration. Moreover, enforced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration. These findings not only reveal a novel mechanism showing that miR-200b inhibits tumor cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop, also suggest that deregulation of miR-200b, Wnt5b and PKCα plays an important role in arsenic carcinogenesis. Citation Format: Zhishan Wang, Brock Humphries, Chengfeng Yang. MicroRNA-200B inhibits arsenic-transformed cell migration by targeting protein kinase Cα signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3108. doi:10.1158/1538-7445.AM2015-3108

MicroRNA-106b inhibits osteoclastogenesis and osteolysis by targeting RANKL in giant cell tumor of bone.

Giant cell tumor (GCT) of bone consists of three major cell types: giant cells, monocytic cells, and stromal cells. From microarray analysis, we found that miR-106b was down-regulated in GCT clinical samples and further determined by fluorescence in situ hybridization. In addition, the expression of novel potential target of miR-106b, RANKL, was elevated in GCT along with previously determined targets in other tumors such as IL-8, MMP2 and TWIST. In a RANKL 3'UTR luciferase reporter assays, agomiR-106b repressed the luciferase activity and the effect was eliminated when the targeting site in the reporter was mutated, suggesting a direct regulation of miR-106b on RANKL mRNA. Moreover, overexpression of miR-106b in GCTSCs through TALEN-mediated site-specific knockin clearly inhibited osteoclastogenesis and osteolysis. By grafting the GCT onto the chick CAM, we confirmed the inhibitory effect of miR-106b on RANKL expression and giant cell formation. Furthermore, in an OVX mouse model, silencing of miR-106b increased RANKL protein expression and promoted bone resorption, while up-regulation of miR-106b inhibited bone resorption. These results suggest that miR-106b is a novel suppressor of osteolysis by targeting RANKL and some other cytokines, which indicates that miR-106b may be a potential therapeutic target for the treatment of GCT.