PURPOSE: Reduction-oxidation reaction homeostasis is vital for regulating inflammatory conditions and its dysregulation may affect the pathogenesis of chronic airway inflammatory diseases such as asthma. Peroxiredoxin-6, an important intracellular anti-oxidant molecule, is reported to be highly expressed in the airways and lungs. The aim of this study was to analyze the expression pattern of peroxiredoxin-6 in the peripheral blood mononuclear cells (PBMCs) of asthmatic patients and in bronchial epithelial cells (BECs).METHODS: The expression levels and modifications of peroxiredoxin-6 were evaluated in PBMCs from 22 asthmatic patients. Phosphorylated and acetylated peroxiredoxin-6 in hydrogen peroxide-treated human BECs was detected using immunoprecipitation analysis. The expression level of peroxiredoxin-6 was also investigated in BECs treated with hydrogen peroxide. Cycloheximide and proteasome inhibitors were used to determine whether peroxiredoxin-6 is degraded by proteasomes.RESULTS: Peroxiredoxin-6 expression was significantly reduced in the PBMCs of asthmatic patients compared to control subjects. Distinct modification patterns for peroxiredoxin-6 were observed in the PBMCs of asthmatic patients using 2-dimensional-electrophoresis. The levels of phosphorylated serine and acetylated lysine in peroxiredoxin-6 were significantly increased in the BECs following hydrogen peroxide treatment. The level of peroxiredoxin-6 expression was reduced in hydrogen peroxide-stimulated BECs, presumably due to proteasomes.CONCLUSIONS: The expression of peroxiredoxin-6, which is down-regulated in the immune cells of asthmatic patients and BECs, can be modified by oxidative stress. This phenomenon may have an effect on asthmatic airway inflammation.
Asthma ; Cycloheximide ; Epithelial Cells ; Homeostasis ; Humans ; Hydrogen ; Hydrogen Peroxide ; Immunoprecipitation ; Inflammation ; Lung ; Lysine ; Oxidative Stress ; Proteasome Inhibitors ; Protein Processing, Post-Translational ; Serine

assay was used for miR-21 expression visualization in lymphoma tissues. Western blot was used for determination of VHL protein, Ki-67, caspase-3, and cleaved caspase-3 levels. Dual-luciferase reporter assay and RNA immunoprecipitation assay were employed to confirm the direct target of miR-21. MTT assay, flow cytometric analysis, and transwell assay were used to evaluate cell proliferation, apoptosis, and migration and invasion capacities, respectively.RESULTS: Curcumin repressed the proliferation, migration, and invasion abilities and promoted apoptosis in SU-DHL-8 cells. Curcumin inhibited miR-21 expression and curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis effects by miR-21 in SU-DHL-8 cells. VHL was a direct target of miR-21. Moreover, curcumin exerted its regulatory effects on SU-DHL-8 cells by VHL.CONCLUSION: Curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis functions, at least partly, by repressing miR-21 and regulating VHL expression in DLBCL cell line. Our findings provided a possible molecular mechanism of curcumin-mediated anti-cancer effect.]]>
Apoptosis ; B-Lymphocytes ; Blotting, Western ; Carcinogenesis ; Caspase 3 ; Cell Line ; Cell Proliferation ; Curcumin ; Humans ; Immunoprecipitation ; In Situ Hybridization ; Lymphoma ; Lymphoma, B-Cell ; MicroRNAs ; Real-Time Polymerase Chain Reaction ; RNA ; RNA, Messenger

BACKGROUND: Increased expression of MDR1 gene is one of the major mechanisms responsible for multidrug resistance in cancer cells. Two alternative promoters, upstream and downstream, are responsible for transcription of MDR1 gene in the human. However, the molecular mechanism regarding the transactivation of MDR1 upstream promoter (USP) has not been determined. METHODS: Dual-luciferase reporter gene assays were used to assess the effect of Nkx-2.5 on MDR1 USP activity using reporter plasmids for human MDR1 USP and its mutants. MDR1 mRNA level was examined by quantitative real-time PCR. The direct binding of Nkx-2.5 to the USP of MDR1 was evaluated by promoter enzyme immunoassays and chromatin immunoprecipitation assays.
Breast Neoplasms ; Breast ; Chromatin Immunoprecipitation ; Drug Resistance, Multiple ; Genes, Reporter ; Humans ; Immunoassay ; Immunoenzyme Techniques ; Phenotype ; Plasmids ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Transcriptional Activation

PURPOSE: Long noncoding RNA 00703 (LINC00703) was found originating from a region downstream of Kruppel-like factor 6 (KLF6) gene, having 2 binding sites for miR-181a. Since KLF6 has been reported as a target of miR-181a in gastric cancer (GC), this study aims to investigate whether LINC00703 regulates the miR-181a/KLF6 axis and plays a functional role in GC pathogenesis.MATERIALS AND METHODS: GC tissues, cell lines, and nude mice were included in this study. RNA binding protein immunoprecipitation (RIP) and pull-down assays were used to evaluate interaction between LINC00703 and miR-181a. Quantitative real-time polymerase chain reaction and western blot were applied for analysis of gene expression at the transcriptional and protein levels. A nude xenograft mouse model was used to determine LINC00703 function in vivo.RESULTS: We revealed that LINC00703 competitively interacts with miR-181a to regulate KLF6. Overexpression of LINC00703 inhibited cell proliferation, migration/invasion, but promoted apoptosis in vitro, and arrested tumor growth in vivo. LINC00703 expression was found to be decreased in GC tissues, which was positively correlated with KLF6, but negatively with the miR-181a levels.CONCLUSIONS: LINC00703 may have an anti-cancer function via modulation of the miR-181a/KLF6 axis. This study also provides a new potential diagnostic marker and therapeutic target for GC treatment.
Animals ; Apoptosis ; Binding Sites ; Blotting, Western ; Cell Line ; Cell Proliferation ; Gene Expression ; Heterografts ; Immunoprecipitation ; In Vitro Techniques ; Mice ; Mice, Nude ; Real-Time Polymerase Chain Reaction ; RNA, Long Noncoding ; RNA-Binding Proteins ; Stomach Neoplasms

PURPOSE: This study was undertaken to explore how miR-206 represses osteosarcoma (OS) development. MATERIALS AND METHODS: Expression levels of miR-206, PAX3, and MET mRNA were explored in paired OS and adjacent tissue specimens. A patient-derived OS cell line was established. miR-206 overexpression and knockdown were achieved by lentiviral transduction. PAX3 and MET overexpression were achieved by plasmid transfection. Treatment with hepatocyte growth factor (HGF) was utilized to activate c-Met receptor. Associations between miR-206 and PAX3 or MET mRNA in OS cells were verified by AGO2-RNA immunoprecipitation assay and miRNA pulldown assay. OS cell malignancy was evaluated in vitro by cell proliferation, metastasis, and apoptosis assays. PAX3 and MET gene expression in OS cells was assayed by RT-qPCR and Western blot. Activation of PI3K-AKT and MAPK-ERK in OS cells were assayed by evaluating Akt1 Ser473 phosphorylation and total threonine phosphorylation of Erk1/2, respectively. RESULTS: Expression levels of miR-206 were significantly decreased in OS tissue specimens, compared to adjacent counterparts, and were inversely correlated with expression of PAX3 and MET mRNA. miR-206 directly interacted with PAX3 and MET mRNA in OS cells. miR-206 overexpression significantly reduced PAX3 and MET gene expression in OS cells in vitro, resulting in significant decreases in Akt1 and Erk1/2 activation, cell proliferation, and metastasis, as well as increases in cell apoptosis, while miR-206 knockdown showed the opposite effects. The effects of miR-206 overexpression on OS cells were reversed by PAX3 or MET overexpression, but only partially attenuated by HGF treatment. CONCLUSION: miR-206 reduces OS cell malignancy in vitro by targeting PAX3 and MET gene expression.
Apoptosis ; Blotting, Western ; Cell Line ; Cell Proliferation ; Gene Expression ; Hepatocyte Growth Factor ; Immunoprecipitation ; In Vitro Techniques ; MicroRNAs ; Neoplasm Metastasis ; Osteosarcoma ; Phosphorylation ; Plasmids ; RNA, Messenger ; Threonine ; Transfection

PURPOSE: Breast cancer (BC) is one of the most common malignant tumors, affecting a significant number of women worldwide. MicroRNAs (miRNAs) have been reported to play important roles in tumorigenesis. The aim of this study was to determine the roles of miR-182-5p in BC progression. MATERIALS AND METHODS: The expressions of miR-182-5p and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were measured in BC tissues and cells by quantitative real-time polymerase chain reaction or Western blot. Cell proliferation and invasion were detected by cell counting kit-8 assay and trans-well assay, respectively. The interaction between miR-182-5p and PTEN was probed by bioinformatics analysis, luciferase activity, and RNA immunoprecipitation. A murine xenograft model was established to investigate the role of miR-182-5p in BC progression in vivo. RESULTS: An abundance of miR-182-5p was noted in BC tissues and cells. High expression of miR-182-5p was associated with poor survival. Abrogation of miR-182-5p inhibited cell proliferation and invasion in BC cells. Interestingly, PTEN was indicated as a target of miR-182-5p, and its restoration reversed miR-182-5p-mediated promotion of proliferation and invasion of BC cells. Moreover, depletion of miR-182-5p suppressed tumor growth via up-regulating PTEN expression in the murine xenograft model. CONCLUSION: MiR-182-5p exhaustion blocked cell proliferation and invasion by regulating PTEN expression, providing a novel therapeutic avenue for treatment of BC.
Blotting, Western ; Breast Neoplasms ; Breast ; Carcinogenesis ; Cell Count ; Cell Proliferation ; Chromosomes, Human, Pair 10 ; Computational Biology ; Female ; Heterografts ; Humans ; Immunoprecipitation ; Luciferases ; MicroRNAs ; Real-Time Polymerase Chain Reaction ; RNA

PURPOSE: Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been deemed an oncogene in many human cancers. However, the underlying mechanism of NEAT1 in nasopharyngeal carcinoma (NPC) progression remains largely unclear. MATERIALS AND METHODS: Quantitative real-time PCR assay was performed to assess the expression of NEAT1 and miR-34a-5p in NPC tissues and cells. Western blot analysis was used to observe cell epithelial to mesenchymal transition (EMT) and the activation of Wnt/β-catenin signaling in 5-8F cells. MiRNA directly interacting with NEAT1 were verified by dual-luciferase reporter assay and RNA immunoprecipitation. Cell proliferation ability was determined by CCK-8 assay, and cell migration and invasion capacities were assessed by transwell assays. An animal model was used to investigate the regulatory effect of NEAT1 on tumor growth in vivo. RESULTS: Our data revealed that NEAT1 is upregulated, while miR-34a-5p is downregulated in NPC tissues and cell lines. NEAT1 knockdown repressed tumor growth in vitro and in vivo. Additionally, we discovered that NEAT1 directly binds to miR-34a-5p and suppresses miR-34a-5p expression. Moreover, NEAT1 knockdown exerted suppression effects on cell proliferation, migration, invasion, and EMT by miR-34a-5p. NEAT1 knockdown blocked Wnt/β-catenin signaling via miR-34a-5p. CONCLUSION: Our study demonstrated that NEAT1 targets miR-34a-5p at least partly to drive NPC progression by regulating Wnt/β-catenin signaling, suggesting a potential therapeutic target for NPC.
Blotting, Western ; Cell Line ; Cell Movement ; Cell Proliferation ; Humans ; Immunoprecipitation ; In Vitro Techniques ; MicroRNAs ; Models, Animal ; Oncogenes ; Real-Time Polymerase Chain Reaction ; RNA ; RNA, Long Noncoding ; Sincalide

5-HT₆ receptor (5-HT₆R) is implicated in cognitive dysfunction, mood disorder, psychosis, and eating disorders. However, despite its significant role in regulating the brain functions, regulation of 5-HT₆R at the molecular level is poorly understood. Here, using yeast two-hybrid assay, we found that human 5-HT₆R directly binds to neuro-oncological ventral antigen 1 (Nova-1), a brain-enriched splicing regulator. The interaction between 5-HT₆R and Nova-1 was confirmed using GST pull-down and co-immunoprecipitation assays in cell lines and rat brain. The splicing activity of Nova-1 was decreased upon overexpression of 5-HT₆R, which was examined by detecting the spliced intermediates of gonadotropin-releasing hormone (GnRH), a known pre-mRNA target of Nova-1, using RT-PCR. In addition, overexpression of 5-HT₆R induced the translocation of Nova-1 from the nucleus to cytoplasm, resulting in the reduced splicing activity of Nova-1. In contrast, overexpression of Nova-1 reduced the activity and the total protein levels of 5-HT₆R. Taken together, these results indicate that when the expression levels of 5-HT₆R or Nova-1 protein are not properly regulated, it may also deteriorate the function of the other.
Animals ; Brain ; Cell Line ; Cytoplasm ; Eating ; Gonadotropin-Releasing Hormone ; Humans ; Immunoprecipitation ; Mood Disorders ; Psychotic Disorders ; Rats ; RNA Precursors ; RNA-Binding Proteins ; Serotonin ; Two-Hybrid System Techniques

Suppressor of Variegation 3–9 Homolog 2 (SUV39H2) methylates the lysine 9 residue of histone H3 and induces heterochromatin formation, resulting in transcriptional repression or silencing of target genes. SUV39H1 and SUV39H2 have a role in embryonic development, and SUV39H1 was shown to suppress cell cycle progression associated with Rb. However, the function of human SUV39H2 has not been extensively studied. We observed that forced expression of SUV39H2 decreased cell proliferation by inducing G1 cell cycle arrest. In addition, SUV39H2 was degraded through the ubiquitin-proteasomal pathway. Using yeast two-hybrid screening to address the degradation mechanism and function of SUV39H2, we identified translationally controlled tumor protein (TCTP) as an SUV39H2-interacting molecule. Mapping of the interacting regions indicated that the N-terminal 60 amino acids (aa) of full-length SUV39H2 and the C-terminus of TCTP (120–172 aa) were critical for binding. The interaction of SUV39H2 and TCTP was further confirmed by co-immunoprecipitation and immunofluorescence staining for colocalization. Moreover, depletion of TCTP by RNAi led to up-regulation of SUV39H2 protein, while TCTP overexpression reduced SUV39H2 protein level. The half-life of SUV39H2 protein was significantly extended upon TCTP depletion. These results clearly indicate that TCTP negatively regulates the expression of SUV39H2 post-translationally. Furthermore, SUV39H2 induced apoptotic cell death in TCTP-knockdown cells. Taken together, we identified SUV39H2, as a novel target protein of TCTP and demonstrated that SUV39H2 regulates cell proliferation of lung cancer cells.
Amino Acids ; Apoptosis ; Carrier Proteins ; Cell Cycle ; Cell Death ; Cell Proliferation ; Embryonic Development ; Female ; Fluorescent Antibody Technique ; G1 Phase Cell Cycle Checkpoints ; Half-Life ; Heterochromatin ; Histones ; Humans ; Immunoprecipitation ; Lung Neoplasms ; Lysine ; Mass Screening ; Pregnancy ; Repression, Psychology ; RNA Interference ; Up-Regulation ; Yeasts

PURPOSE: It is well documented that natural killer (NK) cytotoxicity against hepatocellular carcinoma (HCC) cells is impaired in HCC, which might account for the failure of anti-tumor immune response. miRNAs are considered as important regulators for the development and functions of NK cells. However, the entire role of miR-506 in NK cells remains far from being addressed. MATERIALS AND METHODS: The expressions of miR-506 and signal transducer and activator of transcription 3 (STAT3) mRNA in primary NK cells from HCC patients and healthy controls were detected by quantitative real-time PCR. NK cell cytotoxicity was assessed by CFSE/7AAD cytotoxicity assay and lactate dehydrogenase assay. Luciferase reporter assay, RNA immunoprecipitation assay, and western blot were conducted to confirm the interaction between miR-506 and STAT3. RESULTS: miR-506 expression was downregulated and STAT3 mRNA was upregulated in primary NK cells from HCC patients. Primary NK cells from HCC patients showed remarkably reduced cytotoxicity against SMMC7721 or HepG2 cells. NK cell cytotoxicity was positively correlated with miR-506 expression and negatively correlated with STAT3 mRNA expression. Additionally, miR-506 overexpression enhanced NK cell cytotoxicity against HCC cells, while miR-506 inhibitor showed the reverse effect. Moreover, miR-506 could suppress STAT3 expression by directly targeting 3′-untranslated regions of STAT3. A negative correlation between miR-506 and STAT3 mRNA expression in HCC patients was observed. Mechanistically, overexpressing STAT3 greatly reversed miR-506-mediated promotion of NK cell cytotoxicity against HCC cells. CONCLUSION: miR-506 enhanced NK cell cytotoxicity against HCC cells by targeting STAT3, suggesting that modulating miR-506 expression maybe a promising approach for enhancing NK cell-based antitumor therapies.
Blotting, Western ; Carcinoma, Hepatocellular* ; Hep G2 Cells ; Humans* ; Immunoprecipitation ; Killer Cells, Natural* ; L-Lactate Dehydrogenase ; Luciferases ; MicroRNAs ; Real-Time Polymerase Chain Reaction ; RNA ; RNA, Messenger ; STAT3 Transcription Factor