scholarly journals MiR-590-5p sensitises pancreatic ductal adenocarcinoma cells by blocking autophagy via targeting ATG3

2019 ◽  
Author(s):  
Fazhao Li ◽  
Jun He ◽  
Susun Liu ◽  
Yawei Zhang ◽  
Leping Yang
Keyword(s):  
Cell Lines ◽  
Immunoblot Analysis ◽  
Clonogenic Survival ◽  
Parental Cell ◽  
Luciferase Assay ◽  
Ductal Adenocarcinoma ◽  
Over Expression ◽  
Adenocarcinoma Cells ◽  
Radio Sensitivity

AbstractRadio-resistance is a growing concern in treating patients with pancreatic cancer (PC). Here we investigated the role of miR-590-5p in the radio-resistance of PC cells. We developed radioresistant PC cell lines and followed by microarray analysis and levels of miRs compared to parental cell lines. PC cells were transfected using either miR mimics or inhibitors followed by clonogenic survival assays. We also studied the effect of miR-590-5p on autophagy using electron microscopy and immunoblot analysis. In addition, the luciferase assay was used to identify potential targets. The radio-resistant PC cells exhibited decreased expression of miR-590-5p, with elevated autophagy against the parental cells. The over-expression of miR-590-5p inhibited radiation-mediated autophagy, while inhibitors induced autophagy in PC cells. The up-regulation of miR-590-5p enhanced the radio-sensitivity of PC cells. We confirmed ATG-3 as a target of miR-590-5p, whose levels were unregulated in radio-resistant cells. We also found that levels of ATG-3 were associated with autophagy. Expression of miR-590-5p inhibited radiation-mediated autophagy and enhanced the radio-sensitivity of PC cells.

scholarly journals Tumor Suppressor Role of hsa-miR-193a-3p and -5p in Cutaneous Melanoma

2020 ◽  
Vol 21 (17) ◽  
pp. 6183
Author(s):  
Beatrice Polini ◽  
Sara Carpi ◽  
Stefano Doccini ◽  
Valentina Citi ◽  
Alma Martelli ◽  
...  
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Cutaneous Melanoma ◽  
Luciferase Assay ◽  
Strong Increase ◽  
Over Expression ◽  
Functional Features ◽  
Melanoma Patients ◽  
Melanoma Cell Lines

Background: Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet. Methods: After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and –5p transfection in cutaneous melanoma cell lines are investigated. Results: In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results. Conclusions: Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients.

scholarly journals Chandipura virus dysregulates the expression of hsa-miR-21-5p to activate NF-κB in human microglial cells

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Neha Pandey ◽  
Meghana Rastogi ◽  
Sunit K. Singh
Keyword(s):  
Expression Pattern ◽  
Case Fatality ◽  
Microglial Cells ◽  
Luciferase Assay ◽  
Western India ◽  
Cellular Mirnas ◽  
Over Expression ◽  
Chandipura Virus ◽  
Tnf Α

Abstract Background Chandipura virus (CHPV) is a negative single-stranded RNA virus of the Rhabdoviridae family. CHPV infection has been reported in Central and Western India. CHPV causes acute encephalitis with a case fatality rate of 70 % and mostly affects children below 15 years of age. CHPV infection in brain leads to neuronal apoptosis and activation of the microglial cells. The microRNAs (miRNAs) are small endogenous non-coding RNA that regulate the gene expression. Viral infections perturb the expression pattern of cellular miRNAs, which may in turn affect the expression pattern of downstream genes. This study aims to investigate hsa-miR-21-5p mediated regulation of PTEN, AKT, NF-ĸBp65, IL-6, TNF-α, and IL-1β, in human microglial cells during CHPV infection. Methods To understand the role of hsa-miR-21-5p in CHPV infection, the human microglial cells were infected with CHPV (MOI-0.1). Real-time PCR, western blotting, Luciferase assay, over-expression and knockdown techniques were used to understand the role of hsa-miR-21-5p in the regulation of PTEN, AKT and, NF-ĸBp65, IL-6, TNF-α, and IL-1β in this study. Results The hsa-miR-21-5p was found to be upregulated during CHPV infection in human microglial cells. This led to the downregulation of PTEN which promoted the phosphorylation of AKT and NF-ĸBp65. Over-expression of hsa-miR-21-5p led to the decreased expression of PTEN and promoted further phosphorylation of AKT and NF-ĸBp65 in human microglial cells. However, the inhibition of hsa-miR-21-5p using hsa-miR-21-5p inhibitor restored the expression. Conclusions This study supports the role of hsa-miR-21-5p in the regulation of pro-inflammatory genes in CHPV infected human microglial cells.

miR-132 is up-regulated in polycystic ovarian syndrome and inhibits granulosa cells proliferation via targeting Foxa1

2020 ◽  
Author(s):  
Xiangrong Cui ◽  
Xuan Jing ◽  
Junfen Liu ◽  
Meiqin Yan ◽  
Xingyu Bi ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Polycystic Ovary ◽  
Tumor Cell Line ◽  
Cell Counting ◽  
Luciferase Assay ◽  
Polycystic Ovaries ◽  
Aberrant Expression ◽  
Over Expression ◽  
Ovulatory Dysfunction

Abstract Background: Polycystic ovary syndrome (PCOS) is one of the most common endocrine metabolic disorders characterized by hyperandrogenism, polycystic ovaries and ovulatory dysfunction. Several studies have suggested that the aberrant expression of miRNAs serves an important role in the pathogenesis of PCOS, though the role and underling mechanism of microRNA-132 (miR-132) in the development of PCOS remain unclear. Methods: The expression of miR-132 in granulosa cells (GCs) derived from 26 PCOS patients and 30 healthy controls was detected through RT-qPCR. And the apoptosis levels of granulosa cells were measured by TUNEL.Granulosa-like tumor cell line (KGN) was cultured for cell counting kit-8 (CCK-8) was assays after over-expression of miR-132 or knockdown TargetScan was applied to analysis the potential targets of miR-132, which was further verified by luciferase assay, RT-qPCR and western blot. Results: The expression of miR-132 was declined in granulosa cells of PCOS patients. Meanwhile, the significantly increased apoptotic nuclei were present GCs of PCOS patients. Furthermore, over-expressed of miR-132 inhibited the proliferation of KCN cells. In addition, our results verified that miR-132 directly targeted Foxa1, knockdown of which suppressed KGN cells proliferation. Conclusion: Our results revealed that miR-132 inhibits the cell viability and induces apoptosis by directly interacting with Foxa1, indicating a role of miR-132 to be a potential target in the PCOS patients.

Abstract 271: The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells

2019 ◽  
Author(s):  
Charles R. Dunlop ◽  
Yann Wallez ◽  
Sandra Bernaldo de Quirós Fernández ◽  
Saadia A. Karim ◽  
Alan Lau ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Ductal Adenocarcinoma ◽  
Adenocarcinoma Cells

A Novel Role For Histone Deacetylase 11 (HDAC11) As a Regulator Of Neutrophil Function and Differentiation In Normal and Malignant Hematopoesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2267-2267
Author(s):  
Eva Sahakian ◽  
John Powers ◽  
Jie Chen ◽  
Allison Distler ◽  
Jennifer Rock-Klotz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cytokine Production ◽  
Hematopoietic Cells ◽  
Negative Regulator ◽  
Over Expression

Abstract Histone Deacetylase 11 (HDAC11) is the newest member of the HDAC family of enzymes, which we have previously reported to function as a negative regulator of IL-10 expression in macrophages and dendritic cells. Thus far, its role in other hematopoietic cells has not been completely elucidated. We hereby report for the first time a lineage-restricted over-expression of HDAC11 in neutrophils, committed neutrophil precursors and myeloid leukemias exhibiting neutrophilic differentiation demonstrating a novel physiological role of HDAC11 as a negative regulator of neutrophil cytokine production. Leukocyte subpopulations from murine bone marrow and spleen were flow-sorted and analyzed by qRT-PCR for HDAC11 mRNA, revealing a higher level of mRNA expression on neutrophils and promyelocytes, as compared to monocytes and lymphoid subsets. Similarly, sorted human peripheral blood leukocytes from normal donors, showed higher levels of HDAC11 mRNA in neutrophils, as compared to monocytes. To further investigate the transcriptional activity of HDAC11 in myeloid and lymphoid cells, we utilized a HDAC11 promoter-driven eGFP reporter mice, where eGFP expression indicates HDAC11 transcription (Heintz, N Nat. Rev. Neuroscience 2001). Using multiparametric flow cytometry with lineage-specific markers on this mouse model, we confirmed a marked over-expression of HDAC11 on neutrophils, compared to other subpopulations including monocytes, B-cell, T-cells, NK cells and plasma cells. Furthermore, analysis of bone marrow hematopoietic cells revealed a swift over-expression of HDAC11 at the promyelocyte stage of neutrophil differentiation, with low to undetectable expression in upstream uncommitted common myeloid progenitors and lineage-unrelated monocytic precursors. To study whether this lineage-specific overexpression applies to malignant processes, we studied human cell lines and found overt overexpression of HDAC11 in the acute promyelocytic leukemia cell line NB4, as compared to the myeloblastic cell line Kasumi and two monocyte/macrophage cell lines U937 and THP1. Moreover, in-vitro maturation of the differentiation-inducible myeloid cell line HL60 demonstrated a marked increase in HDAC11 mRNA, paralleling the acquisition of nuclear segmentation characteristic of neutrophil maturation. In order to investigate the physiologic role of HDAC11 overexpression on neutrophils, we utilized a model of germline-HDAC11KO mice. Surprisingly, highly purified neutrophils lacking HDAC11 showed an overt overproduction of TNF-alpha and IL-6 upon stimulation with LPS, as compared to their wild type counterparts. We hereby report a previously un-described lineage-specific over-expression of HDAC11 in neutrophils and its precursors, which actively functions as a physiological repressor of cytokine production and possibly involved in their regulation. Given the predominance of neutrophils which account for 70% of leukocytes in the peripheral blood, and their pivotal role in the first line of defense, results highlight a novel mechanism for HDAC11, as a key regulator and modulator of neutrophil cytokine production with potential implications for autoimmunity, inflammation, and infection. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human Drug Efflux Transporter ABCC5 Confers Sensitivity and Acquired Resistance to Pemetrexed in Breast Cancer

2020 ◽  
Author(s):  
Jihui Chen ◽  
Zhipeng Wang ◽  
Shouhong Gao ◽  
Kejin Wu ◽  
Fang Bai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Abc Transporters ◽  
Cancer Cell Lines ◽  
Control Group ◽  
Over Expression ◽  
Mcf 7

Abstract AimPemetrexed, a new generation antifolate drug, is approved for the treatment for locally advanced or metastatic breast cancer, but factors affecting the efficacy and resistance of it have yet to be fully explicit. ATP-binding cassette transporters have been reported as prognostic and adverse effects predictors of many xenobiotics. This study was designed to explore whether ABC transporters affect pemetrexed resistance and may contribute to treatment regimen optimization for breast cancer.MethodsFirstly, the expression of ABC transporters family members was measured in cell lines, thereafter examined the potential role of ABC transporter in conferring resistance to pemetrexed in primary cancer cell lines isolated from 34 breast cancer patients, and then the role of ABCC5 in mediating transport of pemetrexed and apoptosis pathway in MCF-7 cell lines was assessed. Finally, the functions of ABCC5 on therapeutic effect of pemetrexed was evaluated in breast cancer bearing mice.ResultsThe expressions of ABCC2, ABCC4, ABCC5 and ABCG2 were significantly increased in pan-resistance cell lines, and the ABCC5, the most obvious one, was 5.21 times higher than that of the control group. The expression of ABCC5 was inversely correlated with sensitivity (IC50) of pemetrexed (r = 0.741; p<0.010) in breast cancer cell lines from 34 patients. Further, we found expression of ABCC5 influenced the efflux and cytotoxicity of pemetrexed in MCF-7 cell line, and the IC50 were 0.06 μg/ml and 0.20 μg/ml in ABCC5 knock-down and over-expression cells, respectively. In vivo study, we found ABCC5 affected sensitivity of pemetrexed in breast cancer bearing mice, and the tumor volume was much larger in ABCC5 over-expression group than that in control group (2.7 folds vs 1.2 folds).ConclusionsOur results indicated ABCC5 was associated with pemetrexed sensitivity and resistance in vitro and in vivo, and may be a biomarker for regimen optimization of pemetrexed in breast cancer treatment.

scholarly journals Circular RNA hsa_circ_0001829 promotes gastric cancer progression through miR-155-5p/SMAD2 axis

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Qiuling Niu ◽  
Zhijie Dong ◽  
Min Liang ◽  
Yuanwei Luo ◽  
Hai Lin ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Lines ◽  
Cancer Progression ◽  
Cell Cycle Progression ◽  
Luciferase Assay ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Gain Of Function

Abstract Background Accumulating evidences have shown that circular RNAs (circRNAs) play important roles in regulating the pathogenesis of cancer. However, the role of circRNAs in gastric cancer (GC) remains largely unclear. Methods In this study, we identified a novel upregulated circRNA, hsa_circ_0001829, in chemically induced malignant transformed human gastric epithelial cells using RNA-seq. Subsequent qRT-PCR and ISH assays were performed to detect the expression level of hsa_circ_0001829 in GC cell lines and tissues. Functional roles of hsa_circ_0001829 in GC were then explored by loss- and gain-of- function assays. Bioinformatic prediction and luciferase assay were used to investigate potential mechanisms of hsa_circ_0001829. Finally, the mice xenograft and metastasis models were constructed to assess the function of hsa_circ_0001829 in vivo. Results We found that hsa_circ_0001829 was significantly upregulated in GC tissues and cell lines. Loss- and gain-of- function assays showed that hsa_circ_0001829 promotes GC cells proliferation, migration and invasion, and the affected cell cycle progression and apoptosis rates may account for the effect of hsa_circ_0001829 on GC proliferation. In addition, bioinformatic prediction and luciferase assay showed that hsa_circ_0001829 acts as a molecular sponge for miR-155-5p and that SMAD2 was a target gene of miR-155-5p; moreover, hsa_circ_0001829 sponges miR-155-5p to regulate SMAD2 expression and hsa_circ_0001829 promotes GC progression through the miR-155-5p–SMAD2 pathway. Finally, suppression of hsa_circ_0001829 expression inhibited tumor growth and aggressiveness in vivo. Conclusion Taken together, our findings firstly demonstrated a novel oncogenic role of hsa_circ_0001829 in GC progression through miR-155-5p–SMAD2 axis, and our study may offer novel biomarkers and therapeutic targets for GC.

Abstract 271: The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells

2019 ◽  
Author(s):  
Charles R. Dunlop ◽  
Yann Wallez ◽  
Sandra Bernaldo de Quirós Fernández ◽  
Saadia A. Karim ◽  
Alan Lau ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Ductal Adenocarcinoma ◽  
Adenocarcinoma Cells

NPM-ALK Oncogene Upregulates the Expression of Inducible Nitric Oxide Synthase in T-Cell Lymphoma Through STAT3/MicroRNA-26a-Dependent Mechanism

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1383-1383
Author(s):  
Haifeng Zhu ◽  
Hesham M. Amin
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Lymphoma Cell ◽  
Luciferase Assay ◽  
Inos Mrna ◽  
Inos Protein

Abstract Abstract 1383 The oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is aberrantly expressed in a subset of T-cell anaplastic large-cell lymphoma tumors. NPM-ALK promotes cellular survival and transformation, and initiates lymphomagenesis. To induce its effects, NPM-ALK interacts with a comprehensive network of oncogenes and tumor suppressor genes. Previous studies have identified upstream and downstream members of this network that modulate the effects of NPM-ALK. However, the exact mechanisms by which NPM-ALK induces its effects are not completely identified. Nitric oxide (NO) is a gaseous molecule and a highly active free radical. It plays critical, yet versatile, roles in physiological cellular functions including survival, adhesion, migration, and angiogenesis. It also has important contributions to tumor progression and metastasis. NO is generated from L-arginine via 3 distinct isoforms of the enzyme NO synthase including the inducible form (iNOS). The expression and role of iNOS in NPM-ALK-expressing T-cell lymphoma is not known. We found that iNOS mRNA and protein are expressed in the NPM-ALK-expressing T-cell lymphoma cell lines Karpas 299, DEL, and SR-786. In agreement with a functional role of iNOS in these cells, the selective iNOS inhibitor 1400W and the NO scavenger CPITO decreased the proliferation of the NPM-ALK-expressing T-cell lymphoma cell lines (p < 0.05). In addition, the NO donor SNAP recovered the decrease in the proliferation of these cell lines after treatment with the ALK inhibitors TAE684 and PF-2341066 (p < 0.05). Interestingly, iNOS protein was totally absent in normal human T lymphocytes in spite of a notable increase in iNOS mRNA. These observations suggested that the downregulation of iNOS protein in T lymphocytes vs. its high levels in the NPM-ALK-expressing T-cell lymphoma cell lines might occur posttranscriptionally. Because microRNA are major posttranscriptional regulators of protein expression, we set to analyze possible aberrancies in microRNA. First, we performed an array study comparing the expression of microRNA in T lymphocytes vs. NPM-ALK-expressing T cell lymphoma cell lines. Three web-based algorithms [TargetScan (http:// genes.mit.edu/targetscan/), miRanda (http:// www.microrna.org/microrna/home.do), and PicTar (http:// pictar.mdc-berlin.de/] identified microRNA-26a (miR-26a) to potentially bind with iNOS 3'-UTR. Importantly, statistical analysis of the array data showed that the expression of miR-26a is much more pronounced in T lymphocytes in comparison with the lymphoma cell lines (p < 0.00001), and real-time qPCR further confirmed these results. To examine the functional interactions between miR-26a and iNOS 3'-UTR, we performed a luciferase assay in 293T cells after transfection with iNOS 3'-UTR reporter gene. Wild type miR-26a, and not mutated miR-26a, induced a marked decrease in the luciferase activity of iNOS 3'-UTR reporter gene (p < 0.05). We then questioned whether NPM-ALK underlies the aberrancies in the expression of miR-26a and iNOS. Specific targeting of NPM-ALK by siRNA increased miR-26a and simultaneously decreased iNOS protein levels in the lymphoma cell lines. Next, we reasoned to examine how NPM-ALK induces its effects on this system. A transcription factor screening showed that miR-26a gene promoter, which is located within the promoter of a host gene, CTDSPL, contains 2 consensus sequences where it can potentially bind with STAT3, a major downstream target of NPM-ALK. Chromatin immunoprecipitation studies illustrated the binding between STAT3 and CTDSPL promoter. To analyze the functional interactions between STAT3 and miR-26a, a luciferase assay performed in NPM-ALK-expressing T-cell lymphoma cell lines showed that the downregulation of STAT3 by siRNA increased CTDSPL promoter activity. In addition, downregulation of STAT3 protein or the decrease in STAT3 phosphorylation by STAT3 or NPM-ALK siRNA, respectively, increased miR-26a levels and decreased iNOS protein expression in NPM-ALK-expressing T-cell lymphoma cell lines. Our study proposes novel mechanisms by which NPM-ALK contributes to the survival of T-cell lymphoma. We are currently analyzing the expression of miR-26a and iNOS in primary human lymphoma tumors, and examining the effects of transfecting NPM-ALK-expressing T-cell lymphoma cell lines with miR-26a on cell proliferation, adhesion, and migration. Disclosures: No relevant conflicts of interest to declare.

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