LncRNA-ATB promotes TGF-β-induced glioma cells invasion through NF-κB and P38/MAPK pathway

Glioma constitutes the most aggressive primary intracranial malignancy in adults. We previously showed that long noncoding RNA activated by TGF-β (lncRNA-ATB) promoted the glioma cells invasion. However, whether lncRNA-ATB is involved in TGF-β-mediated invasion of glioma cells remains unknown. In this study, quantitative real-time polymerase chain reaction and western blot analysis were used for detecting the mRNA and protein expression of related genes, respectively. Transwell assay was performed to assess the impact of lncRNA-ATB on TGF-β-induced glioma cells migration and invasion. Immunofluorescence staining was utilized to characterize related protein distribution. Results showed that TGF-β upregulated lncRNA-ATB expression in glioma LN-18 and U251 cells. Overexpression of lncRNA-ATB activated nuclear factor-κB (NF-κB) pathway and promoted P65 translocation into the nucleus, thus facilitated glioma cells invasion stimulated by TGF-β. Similarly, lncRNA-ATB markedly enhanced TGF-β-mediated invasion of glioma cells through activation P38 mitogen-activated protein kinase (P38/MAPK) pathway. Moreover, both the NF-κB selected inhibitor pyrrolidinedithiocarbamate ammonium and P38/MAPK specific inhibitor SB203580 partly reversed lncRNA-ATB induced glioma cells invasion mediated by TGF-β. Collectively, this study revealed that lncRNA-ATB promotes TGF-β-induced glioma cell invasion through NF-κB and P38/MAPK pathway and established a detailed framework for understanding the way how lncRNA-ATB performs its function in TGF-β-mediated glioma invasion.     

NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.     

Downregulated circRNA_CDKN1A promotes gallbladder cancer progression through activation of the NF-κB pathway

This study uncovered the potential clinical value and molecular driving mechanisms of circular RNAs (circRNAs) in gallbladder cancer (GBC). Differentially expressed circRNAs in GBC cells were screened by high-throughput sequencing. CircRNA_CDKN1A (circBase ID: hsa_circ_0076194) was knocked out in BGC-SD cells through transfection with sh-circRNA_CDKN1A. Then, proliferation was investigated via CCK8 and EdU assays, apoptosis via flow cytometry, migration via wound healing assays, and invasion via Transwell assays. Bioinformatics analysis of circRNA_CDKN1A-related signaling pathways was performed using MetScape and g:Profiler. Results showed that the knockdown of circRNA_CDKN1A enhanced the proliferation, migration, and invasion of GBC cells and inhibited apoptosis. In addition, knocking out circRNA_CDKN1A promoted GBC cell proliferation and enhanced the dry indices of the OCT4 protein and CD34 expression levels. The knockdown of circRNA_CDKN1A activated the epithelial–mesenchymal transition pathway. Bioinformatics analysis revealed that the biological role of circRNA_CDKN1A in GBC cells involved the NF-κB pathway. LY2409881, which is an NF-κB inhibitor, reversed the effects induced by the knockdown of circRNA_CDKN1A in GBC-SD cells. In summary, the knockdown of circRNA_CDKN1A promoted the progression of GBC by activating the NF-κB signaling pathway. For the first time, this study revealed the mechanism of circRNA_CDKN1A-mediated regulatory action in GBC and identified the newly discovered circRNA_CDKN1A–NF-κB signaling axis as a potentially important candidate for clinical therapy and prognostic diagnosis of GBC.     

TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-β1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-β1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-β1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-β1-stimulated invasion. Our results also indicate that signaling events involved in TGF-β1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.     

TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type (“ricin-like”) lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.     

TWEAK/Fn14 promotes the proliferation and collagen synthesis of rat cardiac fibroblasts via the NF-кB pathway

We wished to elucidate a potential role of the tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible molecule 14 (Fn14) axis in myocardial fibrosis. Stimulation of neonatal rat cardiac fibroblasts (CFs) with TWEAK could increase CFs numbers and collagen synthesis. Conversely, when CFs were pretreated with siRNA against Fn14, induction of cell proliferation and collagen synthesis by TWEAK were inhibited. Pretreatment with TWEAK on CFs induced activation of the nuclear factor-kappaB (NF-кB) pathway and subsequently increased the production of metalloproteinase-9 (MMP-9). Cell treatment with siRNA against Fn14 led to inhibition of the NF-кB pathway. Additionally, after stimulation of cell with ammonium pyrrolidine dithiocarbamate, cell proliferation and collagen synthesis induced by NF-кB and the upregulation of MMP-9 production were inhibited. The present study suggested that the TWEAK/Fn14 axis increased cell proliferation and collagen synthesis by activating the NF-кB pathway and increasing MMP-9 activity. This axis may be important for regulating myocardial fibrosis.     

Id-1 promotes migration and invasion of non-small cell lung cancer cells through activating NF-κB signaling pathway

Background

Numerous studies have shown that Id-1 (Inhibitor of differentiation 1) is upregulated in several cancers and associated with tumor malignant characters. However, the clinical significance and biological role of Id-1 in non-small cell lung cancer (NSCLC) remains unclear.

Methods

We used RT-PCR, Western blot and Immunohistochemistry to measure Id-1 expression in NSCLC tissues and matched adjacent noncancerous tissues. The expression pattern of Id-1 in NSCLC tissues was determined by scoring system of immunohistochemical analysis. The Kaplan-Meier method was used to calculate the survival curve, and log-rank test to determine statistical significance. The Id-1 gene was overexpressed or downreuglated with Lentiviral vectors in NSCLC cells. And, the migration ability of NSCLC cells was tested in a Transwell Boyden Chamber.

Results

We found that Id-1 is generally expressed higher in NSCLC tissues compared with matched adjacent noncancerous tissues. We also found that high Id-1 expression in tumor tissues is significantly correlated with tumor progression and poor survival in NSCLC patients. Furthermore, our experimental data revealed that knockdown of Id-1 significantly suppressed the proliferation, migration and invasion of NSCLC cells, whereas ectopic expression of Id-1 promoted the malignant phenotype of NSCLC cells. Mechanistic study showed that NF-κB signaling pathway contributed to the effects of Id-1 in NSCLC cells. Moreover, blocking the NF-κB pathway significantly inhibited the tumor-promoting actions of Id-1 in NSCLC cells.

Conclusions

We identified a tumorigenic role of Id-1 in NSCLC and provided a novel therapeutic target for NSCLC patients.

     

MTA1 promotes the invasion and migration of pancreatic cancer cells potentially through the HIF-α/VEGF pathway

Abstract

The metastasis-associated gene 1 (MTA1) has previously been recognized as an oncogene, and abnormal MTA1 expression has been related to progression of numerous cancer types to the metastasis stage. However, the function of MTA1 in the regulation of pancreatic cancer progression and metastasis remains unclear. Western blot analysis was adopted to determine the expression of MTA1 in pancreatic cancer tissues and corresponding near normal tissues. Steady clone with MTA1-overexpression and MTA1-inhibitionweregenerated via lentivirus technology in BxPc-3 cells. Transwell assay was carried out for detecting the invasion of pancreatic cancer cells. The migration activity was assessed using the wound scratch assay. The effect of MTA1 in pancreatic cancer was evaluated in the mice xenografts. Western blot analysis was employed to determine the expression of hypoxia inducible factor-α (HIF-α) and vascular endothelial growth factor (VEGF) in vitro and in vivo. We observed that MTA1 overexpression enhanced migration and invasion ability of pancreatic cancer cells in vitro and increased HIF-α and VEGF protein levels in vitro and in vivo. MTA1 inhibition had the opposite effects. MTA1 protein level was positively related to HIF-α and VEGF protein levels. These results indicated that MTA1 potentially promoted pancreatic cancer metastasis via HIF-α/VEGF pathway. This research supplies a new molecular mechanism for MTA1 in the pancreatic cancer progression and metastasis. MTA1 may be an effective therapy target in pancreatic cancer.     

MMP-11 promotes papillary thyroid cell proliferation and invasion via the NF-κB pathway

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer, and its incidence is on the rise. It has been reported that some matrix metalloproteinases (MMPs) are abnormally expressed in PTC and can be used as diagnostic markers. However, few studies have explored the underlying mechanisms by which MMPs promote tumor progression. In this study, we used microarray analysis to compare the variations of gene expression within the PTC cell populations and their adjacent normal tissues and found that MMP-11 was the most differentially expressed MMP. To investigate the role of MMP-11 in the mediation of thyroid cancer cell development, pEnter-MMP-11 plasmid, and MMP-11 small interfering RNA were applied to up- and downregulate MMP-11 expression of in cultured PTC cell lines K1 and BCPAP. The results suggested that the levels of proliferation and migration of cells transfected with MMP-11 siRNA were significantly reduced, while the levels in MMP-11-plasmid-transfected cells were increased. In terms of the mechanism, experimental data showed that the change in cyclin D1 is consistent with MMP-11 expression, which may explain the changes in proliferation. In addition, Western blot assay was conducted to analyze the p65 and activated (phospho-) p65 protein levels concomitant with MMP-11 adjustments. Variations in intracellular MMP-11 significantly altered the amount of phospho-p65 in thyroid cells, while p65 knockdown did not affect MMP-11 expression. These results suggest that MMP-11 is located upstream of p65 and regulates its activity. Interestingly, the data for the Transwell assay suggested that MMP-11 regulatory migration is also associated with the NF-κB p65 signaling pathway. In conclusion, this report describes the important role of MMP-11 in the regulation of thyroid cell proliferation and migration. Mechanistic studies have shown that cyclin D1 and p65 are important mediators in the processes, which provides a new way to study the mechanism of MMPs promoting the progression of thyroid cancer.     

IL-37 inhibits the maturation of dendritic cells through the IL-1R8-TLR4-NF-κB pathway

Mature dendritic cells (DCs) play a pathogenic role in atherosclerosis. Our previous study demonstrated that exogenous interleukin (IL)-37 suppresses the maturation of DCs, induces the T-regulatory (Treg) cell response, and attenuates atherosclerosis in ApoE^−/− mice. The aim of the present study was to explore the molecular mechanism of IL-37 on the maturation of DCs throughout the development of atherosclerosis. The expression of interleukin-1 receptor 8 (IL-1R8), which is a single Ig-domain receptor that was recently found to be pivotal for the extracellular function of IL-37, Toll-like receptor (TLR) 4 and p65, was measured in ApoE^−/− mice and IL-37 transgenic (IL-37tg) ApoE^−/− mice. IL-1R8 was mainly expressed in aortic plaque-infiltrated DCs and at significantly higher levels in IL-37tg atherosclerotic mice, accompanied by lower levels of TLR4 and p65. Furthermore, IL-37 eliminated the maturation of DCs induced by oxidized low-density lipoprotein (oxLDL) and caused marked upregulation of IL-1R8 in vitro and downregulation of TLR4 and p65, which was consistent with the experiments in mice. However, the inhibitory effect of IL-37 on the maturation of DCs in vitro was abolished when IL-37 was used to treat DCs isolated from IL-1R8-deficient and TLR4-deficient mice. Therefore, this study indicated that IL-37 inhibited the maturation of DCs via the IL-1R8-TLR4-NF-κB pathway and attenuated atherosclerosis in ApoE^−/− mice.     

Ring1 promotes the transformation of hepatic progenitor cells into cancer stem cells through the Wnt/β-catenin signaling pathway

The proliferation of hepatic progenitor cells (HPCs) is observed in reactive conditions of the liver and primary liver cancers. Ring1 as a member of polycomb-group proteins which play vital roles in carcinogenesis and stem cell self-renewal was increased in HCC patients and promoted proliferation and survival of cancer cell by degrading p53. However, the mechanisms of Ring1 driving the progression of hepatocarcinogenesis have not been elucidated. In this study, forced expression Ring1 and Ring1 siRNA lentiviral vectors were utilized to stably overexpression and silence Ring1 in HPC cell line (WB-F344), respectively. Our finding indicated that overexpression of Ring1 in HPCs promoted colony formation, cell multiplication, and invasion in vitro, conversely depletion of Ring1 repressed the biological functions of HPCs relative to controls. The expression of β-catenin was upregulated in the HPCs with overexpression of Ring1, and the correlation analysis also showed that β-catenin and Ring1 had a significant correlation in the liver cancer tissues and adjacent tissues. The activation of the Wnt/β-catenin signaling pathway significantly increased the expression of liver cancer stem cells related (LCSCs)-related molecular markers CD90 and EpCAM, which led to the transformation of HPCs into LCSCs. Most importantly, the injection of HPCs with overexpressed Ring1 into the subcutaneous of nude mice leads to the formation of poorly differentiated HCC neoplasm. Our findings elucidate that overexpression of Ring1 the activated Wnt/β-catenin signaling pathway and drove the transformation of HPCs into cancer stem cell-like cells, suggesting Ring1 has extraordinary potential in early diagnosis of HCC.     

Testes-specific protease 50 (TSP50) promotes cell proliferation through the activation of the nuclear factor κB (NF-κB) signalling pathway

TSP50 (testes-specific protease 50) is a testis-specific expression protein, which is expressed abnormally at high levels in breast cancer tissues. This makes it an attractive molecular marker and a potential target for diagnosis and therapy; however, the biological function of TSP50 is still unclear. In the present study, we show that overexpression of TSP50 in CHO (Chinese-hamster ovary) cells markedly increased cell proliferation and colony formation. Mechanistic studies have revealed that TSP50 can enhance the level of TNFα (tumour necrosis factor α)- and PMA-induced NF-κB (nuclear factor κB)-responsive reporter activity, IκB (inhibitor of NF-κB) α degradation and p65 nuclear translocation. In addition, the knockdown of endogenous TSP50 in MDA-MB-231 cells greatly inhibited NF-κB activity. Co-immunoprecipitation studies demonstrated an interaction of TSP50 with the NF-κB-IκBα complex, but not with the IKK (IκB kinase) α/β-IKKγ complex, which suggested that TSP50, as a novel type of protease, promoted the degradation of IκBα proteins by binding to the NF-κB-IκBα complex. Our results also revealed that TSP50 can enhance the expression of NF-κB target genes involved in cell proliferation. Furthermore, overexpression of a dominant-negative IκB mutant that is resistant to proteasome-mediated degradation significantly reversed TSP50-induced cell proliferation, colony formation and tumour formation in nude mice. Taken together, the results of the present study suggest that TSP50 promotes cell proliferation, at least partially, through activation of the NF-κB signalling pathway.     

Construction of NF-κB-targeting RNAi adenovirus vector and the effect of NF-κB pathway on proliferation and apoptosis of vascular endothelial cells

To construct a recombinant adenovirus vector expressing a RNAi for the Nuclear Factor kappa B (NF-κB)/p65 gene and use it to explore the role of the NF-κB pathway on the regulation of proliferation and apoptosis of vascular endothelial cells. A recombinant adenovirus containing a RNAi cassette targeting the p65 gene was constructed, and its silencing effect on p65 was detected by Western blot analysis in ECV304 cells. Expression of the p65 protein in ECV304 cells was efficiently down-regulated by the RNAi adenovirus for more than 6 days. ECV304 cells proliferation and apoptosis were measured using the MTT assay and flow cytometry, respectively. Blocking the NF-κB pathway with the RNAi adenovirus substantially decreased the proliferation of ECV304 cells, but only slightly affected cell apoptosis. We used a NF-κB/p65-targeting RNAi adenovirus to demonstrate the role of the NF-κB pathway in the regulation of ECV304 cell proliferation. This adenovirus may serve as an important tool to study the NF-κB pathway.