Objective: To investigate the regulatory effect of the transcription factor NF-kB1 on the expression of miR-195 in prostate cancer (PCa). METHODS: We analyzed the possibility of NF-kB1 binding to the miR-195 promoter and the expression of NF-kB1 in PCa using the JASPAR and Oncomine databases, respectively, and determined the expressions of NF-kB1 and miR-195 in PCa cells by real-time quantitative PCR after inhibiting the former by interfering RNA targeting NF-kB1. We detected the activity of the luciferase reporter gene after constructing its gene plasmid in the miR-195 promoter region and having it co-transfected with the NF-kB1 plasmid. Then we analyzed the correlation between the expressions of miR-195 and NF-kB1 in the prostate tissue. RESULTS: NF-kB1 was overexpressed in PCa. After inhibition of the expression of NF-kB1, that of miR-195 was increased in PC-3 and DU-145 cell lines, with a negative correlation between the NF-kB1 and miR-195 expressions in the PCa tissue. The results of luciferase reporter gene assay showed direct binding of NF-kB1 to the miR-195 promoter zone. CONCLUSIONS NF-kB1 regulates the expression of miR-195 in prostate cancer.
Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; MicroRNAs/genetics* ; NF-kappa B p50 Subunit/metabolism* ; Promoter Regions, Genetic ; Prostatic Neoplasms/genetics* ; Transcription Factors/metabolism*

Objective: Hyperbaric oxygen treatment (HBOT) has demonstrated efficacy in improving hearing levels of patients with idiopathic sudden sensorineural hearing loss (ISSHL); however, the underlying mechanisms are not well understood. HBOT alleviates the inflammatory response, which is mediated by Toll-like receptor (TLR) 4 and nuclear factor (NF)-κB. In this study we investigated whether HBOT attenuates inflammation in ISHHL patients alteration of TLR4 and NF-κB expression. Methods: ISHHL patients ( = 120) and healthy control subjects ( = 20) were enrolled in this study. Patients were randomly divided into medicine group treated with medicine only ( = 60) and HBO group receiving both HBOT and medicine ( = 60). Audiometric testing was performed pre- and post-treatment. TLR4, NF-кB, and TNF-α expression in peripheral blood of ISSHL patients and healthy control subjects was assessed by ELISA before and after treatment. Results: TLR4, NF-κB, and TNF-α levels were upregulated in ISSHL patients relative to healthy control subjects; the levels were decreased following treatment and were lower in the HBO group than that in the medicine group post-treatment ( < 0.05 and < 0.01). Conclusion HBOT alleviates hearing loss in ISSHL patients by suppressing the inflammatory response induced by TLR4 and NF-κB signaling.
Adolescent ; Adult ; Aged ; China ; Female ; Hearing Loss, Sensorineural ; therapy ; Hearing Loss, Sudden ; therapy ; Humans ; Hyperbaric Oxygenation ; Inflammation ; genetics ; therapy ; Male ; Middle Aged ; NF-kappa B p50 Subunit ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Young Adult

Annexin A2, a multifunctional tumor associated protein, promotes nuclear factor-kappa B (NF-κB) activation by interacting with NF-κB p50 subunit and facilitating its nuclear translocation. Here we demonstrated that two ginsenosides Rg5 (G-Rg5) and Rk1 (G-Rk1), with similar structure, directly bound to Annexin A2 by molecular docking and cellular thermal shift assay. Both Rg5 and Rk1 inhibited the interaction between Annexin A2 and NF-κB p50 subunit, their translocation to nuclear and NF-κB activation. Inhibition of NF-κB by these two ginsenosides decreased the expression of inhibitor of apoptosis proteins (IAPs), leading to caspase activation and apoptosis. Over expression of K302A Annexin A2, a mutant version of Annexin A2, which fails to interact with G-Rg5 and G-Rk1, effectively reduced the NF-κB inhibitory effect and apoptosis induced by G-Rg5 and G-Rk1. In addition, the knockdown of Annexin A2 largely enhanced NF-κB activation and apoptosis induced by the two molecules, indicating that the effects of G-Rg5 and G-Rk1 on NF-κB were mainly mediated by Annexin A2. Taken together, this study for the first time demonstrated that G-Rg5 and G-Rk1 inhibit tumor cell growth by targeting Annexin A2 and NF-κB pathway, and G-Rg5 and G-Rk1 might be promising natural compounds for targeted cancer therapy.
Active Transport, Cell Nucleus ; drug effects ; Annexin A2 ; chemistry ; deficiency ; genetics ; metabolism ; Antineoplastic Agents ; chemistry ; metabolism ; pharmacology ; Apoptosis ; drug effects ; Biological Products ; chemistry ; metabolism ; pharmacology ; Cell Nucleus ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Drug Discovery ; Gene Knockdown Techniques ; Ginsenosides ; chemistry ; Hep G2 Cells ; Humans ; Molecular Docking Simulation ; Molecular Targeted Therapy ; NF-kappa B p50 Subunit ; metabolism ; Protein Conformation

We evaluated the effect of cobalt chloride (CoCl2) on TNF-alpha and IFN-gamma-induced-inflammation and reactive oxygen species (ROS) in renal tubular epithelial cells (HK-2 cells). We treated HK-2 cells with CoCl2 before the administration of TNF-alpha/IFN-gamma. To regulate hemeoxygenase-1 (HO-1) expression, the cells were treated CoCl2 or HO-1 siRNA. CoCl2 reduced the generation of ROS induced by TNF-alpha/IFN-gamma. TNF-alpha/IFN-gamma-treated-cells showed an increase in the nuclear translocation of phosphorylated NF-kappaBp65 protein, the DNA-binding activity of NF-kappaBp50 and NF-kappaB transcriptional activity and a decrease in IkappaBalpha protein expression. These changes were restored by CoCl2. We noted an intense increase in monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) production in TNF-alpha/IFN-gamma-treated cells. We demonstrated that this effect was mediated through NF-kappaB signaling because an NF-kappaB inhibitor significantly reduced MCP-1 and RANTES production. CoCl2 effectively reduced MCP-1 and RANTES production. The expression of HO-1 was increased by CoCl2 and decreased by HO-1 siRNA. However, knockdown of HO-1 by RNA interference did not affect MCP-1 or RANTES production. We suggest that CoCl2 has a protective effect on TNF-alpha/IFN-gamma-induced inflammation through the inhibition of NF-kappaB and ROS in HK-2 cells. However, CoCl2 appears to act in an HO-1-independent manner.
Cell Line ; Chemokine CCL2/metabolism ; Chemokine CCL5/metabolism ; Cobalt/*pharmacology ; Epithelial Cells/cytology/metabolism ; Heme Oxygenase-1/antagonists & inhibitors/genetics/metabolism ; Humans ; *Inflammation ; Interferon-gamma/pharmacology ; Kidney Tubules, Proximal/cytology ; NF-kappa B/antagonists & inhibitors/genetics/*metabolism ; NF-kappa B p50 Subunit/genetics/metabolism ; Oxidative Stress/*drug effects ; Phosphorylation ; Protein Binding ; RNA Interference ; RNA, Small Interfering/metabolism ; Transcription Factor RelA/metabolism ; Tumor Necrosis Factor-alpha/pharmacology

This study aims to express pig nuclear transcription factor-kappaB (NF-kappaB) p65/p50 fusion protein in E. coli Rosetta, and study its impacts on PRRSV proliferation in vitro. The p65 ORF and mature p50 encoding gene were amplified by RT-PCR, the products were cloned into the pET-21a(+) vector, then transformed into Escherichia coli Rosetta, recombinant fusion protein was expressed by IPTG induction, the expressed product was identified by SDS-PAGE and Western-Blot. The purified and re-folded p65/p50 was added to the 2% FBS DMEM, and the cytotoxicity on Marc145 was observed to select the optimum concentration. The effects of optimum concentration of p65/p50 on PRRSV proliferation activity were investigated by detecting PRRSV infection phase in the culture supernatant using real-time FQ-PCR method and drawing PRRSV one-step growth curve. The results showed the p65/p50-pET21a(+) prokaryotic expression vector were successfully constructed , recombinant p50 and p65 fusion protein was expressed abundantly in the form of inclusion body with molecular weight of 70kD, Western-Blot results showed that the rabbit anti-human p50 polyclonal serum, rabbit anti-human p65 purified antibody could bind specifically to p50 and p65 respectively. The optimum concentration of p65/p50 was 0.4 microg/mL. The real-time FQ-PCR results indicated that NF-kappaB p65/p50 could promote CPE appearance and PRRSV proliferation before CPE appeared, and suppress PRRSV proliferation after CPE appeared, and lower the virus titer levels significantly(P < 0.05). These results will provide some new insight of the pathogenic mechanism and treatment strategies of PRRS.
Animals ; Cell Line ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; NF-kappa B p50 Subunit ; genetics ; metabolism ; Porcine Reproductive and Respiratory Syndrome ; metabolism ; Porcine respiratory and reproductive syndrome virus ; genetics ; physiology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Swine ; Transcription Factor RelA ; genetics ; metabolism ; Virus Replication

<b>OBJECTIVEb>To explore how NF-&kappa;B family members regulate maspin expression in prostate cancer cells.

<b>METHODSb>The expression of NF-&kappa;B subunits and maspin was detected by Western blot analysis in prostate cancer DU145, PC-3, and LNCaP cell lines. RNA interference was performed to analyze whether RelB- or RelA-deletion affectes cell death as well as the expression of NF-&kappa;B subunits and maspin. The impact of RelB-silencing in DU145 cells was investigated by flow cytometry. The regulation of RelB on maspin expression in the prostate cancer PC-3 cells was also examined via stable transfection of RelB expression plasmid.

<b>RESULTSb>RelA, p50, RelB, and p52 were constitutively expressed in androgen-independent prostate cancer DU145 and PC-3 cells, while RelB had the highest expression in DU145 cells. Low expression of maspin was detected in LNCaP and DU145 cells, but elevated expression in PC-3 cells. RelB-silencing in DU145 cells by siRNA interference upregulated the endogenous expression of maspin and induced cell apoptosis (13.3±4.2)%. Overexpression of RelB in PC-3 cells inhibited the endogenous expression of maspin. RelA-silecing had no significant influence on the endogenous expression of maspin.

<b>CONCLUSIONSb>The classical and alternative NF-&kappa;B activitions are sustained in androgen-independent prostate cancer cell lines. The expressions of RelB and maspin are inversely correlated in these cancer cells. The expression of RelB negatively regulates the endogenous expression of maspin, then interferes the cell survival. RelA is not involved in the regulation of maspin expression.

Apoptosis ; Cell Line, Tumor ; Gene Silencing ; Humans ; Male ; NF-kappa B ; genetics ; metabolism ; NF-kappa B p50 Subunit ; genetics ; metabolism ; NF-kappa B p52 Subunit ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA, Small Interfering ; genetics ; Serpins ; metabolism ; Transcription Factor RelA ; genetics ; metabolism ; Transcription Factor RelB ; genetics ; metabolism ; Transfection

<b>OBJECTIVEb>To pinpoint angiogenesis- and lymphangiogenesis-related genes in nasopharyngeal carcinoma (NPC).

<b>METHODSb>Based on the reported microarray data which identified 831 differentially expressed genes in NPC tissues and the latest genomic information, we selected 246 genes for analysis with the smallest differential expression threshold of 260. Gene function analysis and network construction was carried out based on literature mining for analysis of the signaling pathways related with angiogenesis and lymphangiogenesis of NPC.

<b>RESULTSb>The 246 genes were related with such keywords as nasopharyngeal carcinoma, EB virus, metastasis, angiogenesis, lymphangiogenesis, and invasion. Particularly, we found that up to 52 genes were associated with angiogenesis (P=0.00001), and 19 genes form 12 related gene pairs (P=0.0042). Twenty-one lymphangiogenesis-related genes were identified (P=0.00001), and 6 of these genes formed a gene network (P=0.0226). Eight genes, including PTGS2, participated in the nuclear factor-&kappa;B (NF-&kappa;B) pathway, which was closely related to angiogenesis in small cell lung cancer (P=7.87E-07). Five genes, including STAT1 and CXCL10, participated in toll-like receptor signaling pathway (P=0.00176).

<b>CONCLUSIONb>PTGS2 and NF-&kappa;B promote angiogenesis of NPC, and the role of toll-like receptor signaling pathway in lymphangiogenesis warrants further investigation.

Carcinoma ; Carcinoma, Squamous Cell ; blood supply ; genetics ; pathology ; Cyclooxygenase 2 ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphangiogenesis ; NF-kappa B p50 Subunit ; metabolism ; Nasopharyngeal Neoplasms ; blood supply ; genetics ; pathology ; Neovascularization, Pathologic ; Oligonucleotide Array Sequence Analysis ; Signal Transduction ; Toll-Like Receptors ; metabolism

The activation of nuclear factor-kappa B1 (NFkappaB1) in cancer cells may confer resistance to ionizing radiation (IR). To enhance the therapeutic efficiency of IR in lung cancer, we screened for microRNAs (miRNAs) that suppress NFkappaB1 and observed their effects on radiosensitivity in a human lung cancer cell line. From time series data of miRNA expression in gamma-irradiated H1299 human lung cancer cells, we found that the expression of miR-9 was inversely correlated with that of NFkappaB1. Overexpression of miR-9 down-regulated the level of NFkappaB1 in H1299 cells, and the surviving fraction of gamma-irradiated cells was decreased. Interestingly, let-7g also suppressed the expression of NFkappaB1, although there was no canonical target site for let-7g in the NFkappaB1 3' untranslated region. From these results, we conclude that the expression of miR-9 and let-7g could enhance the efficiency of radiotherapy for lung cancer treatment through the inhibition of NFkappaB1.
Base Sequence ; Cell Line, Tumor ; Cell Survival/genetics/radiation effects ; Gene Expression Profiling ; *Gene Expression Regulation, Neoplastic/radiation effects ; Humans ; Lung Neoplasms/genetics/metabolism ; MicroRNAs/genetics/*metabolism ; NF-kappa B p50 Subunit/genetics/*metabolism ; Radiation Tolerance/*genetics ; Radiation, Ionizing ; Sequence Alignment

OBJECTIVE: To study the expression and activation of p50 subunit of nuclear factor-kappa B(NF-kappaB) in mucosa of seasonal allergic rhinitis. METHOD: The expression of p50 subunit of NF-kappaB in the mucosa from 16 patients (6 patients with symptom, 10 patients without symptom)and 10 normal subjects were detected by immunohistochemistry. The activation of DNA-binding proteins which was labeled with 32P-radiolabeled oligonucleotide probe for NF-kappaB was detected with electrophoretic mobility shift assays(EMSA) in mucosa. RESULT: The expression of p50 subunit of NF-kappaB was observed in the nasal mucosa of SAR and normal samples. The expression of p50 subunit of NF-kappaB was positive in the cytoplasm and some nuclei of the mucosal epithelia, inflammatory cells, glandular epithelia, and vascular endothelia in nasal mucosa. The Rate of nucleus positive staining of p50 was (41.83 +/- 4.43)% and(37.19 +/- 3.93)% in SAR with symptom and SAR without symptom patients,respectively. There was no significant difference (P > 0.05). The Rate of nucleus positive staining of p50 in nasal mucosa of normal samples was (8.89 +/- 1.32)%. The difference of p50 subunit of NF-kappaB expression between SAR group and normal group was statistically significant (P < 0.01). The DNA-binding proteins activity of samples from patients with seasonal allergic rhinitis was stronger than that in normal subjects (P < 0.05). CONCLUSION p50 subunit of NF-kappaB was activated in healthy nasal mucosa to some extent. The expression and DNA-binding proteins activity of p50 subunit of NF-kappaB was enhanced in seasonal allergic rhinitis. It indicated that p50 subunit of NF-kappaB may be involved in nasal mucosa physiological function and may have an important role in maintaining the chronic inflammation in seasonal allergic rhinitis.
Case-Control Studies ; Humans ; NF-kappa B p50 Subunit ; genetics ; metabolism ; Nasal Mucosa ; metabolism ; Rhinitis, Allergic, Seasonal ; metabolism

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor (TFPI) gene expression through the androgen receptor in endothelial cells. This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-alpha. TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction. To study the cellular mechanism of testosterone's action, nuclear factor-kappa B (NF-kappaB) translocation was confirmed by electrophoretic mobility shift assays. We found that after NF-kappaB was activated by TNF-alpha, TFPI protein levels declined significantly by 37.3% compared with controls (P < 0.001), and the mRNA levels of TFPI also decreased greatly (P < 0.001). A concentration of 30 nmol L(-1) testosterone increased the secretion of TFPI compared with the TNF-alpha-treated group. NF-kappaB DNA-binding activity was significantly suppressed by testosterone (P < 0.05). This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-kappaB activity.
Androgens ; pharmacology ; Cells, Cultured ; Down-Regulation ; drug effects ; Drug Combinations ; Endothelium, Vascular ; drug effects ; metabolism ; Humans ; Infant, Newborn ; Lipoproteins ; genetics ; metabolism ; NF-kappa B p50 Subunit ; antagonists & inhibitors ; genetics ; RNA, Messenger ; metabolism ; Testosterone ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology