Expression Of Tumor Necrosis Factor Receptor-associated Factor 6 Research Articles

Circ_0010729 knockdown protects cardiomyocytes against hypoxic dysfunction via miR-370-3p/TRAF6 axis.

Few studies have addressed the mechanism by which circ_0010729 regulates hypoxia-induced cell injury in cardiovascular diseases. However, its role and its regulatory mechanism in myocardial infarction remain to be explored. Cell viability, cycle, apoptosis, and migration were analyzed using cell counting kit-8 assay, flow cytometry, caspase-3 activity assay kit and transwell assay, respectively. Tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations were examined by enzyme-linked immunosorbent assay. Glucose metabolism was calculated by detecting ATP production, glucose uptake and lactate production. Levels of circ_0010729, miR-370-3p and TNF Receptor Associated Factor 6 (TRAF6) were detected using quantitative real-time polymerase chain reaction or western blot. The direct interaction between circ_0010729 and TRAF6 or miR-370-3p was verified using dual-luciferase reporter assay and RNA immunoprecipitation assay. Under hypoxia condition, cardiomyocytes suffered from cell viability suppression, cell cycle arrest, cell apoptosis promotion, migration reduction, increase of inflammatory factor IL-6 and TNF-α, as well as glycolysis inhibition. Circ_0010729 expression was up-regulated in the cardiomyocytes at different hypoxia-exposed time points. Circ_0010729 knockdown protected cardiomyocytes against hypoxic dysfunction, while circ_0010729 overexpression showed inverse effects. MiR-370-3p was confirmed to directly bind to circ_0010729 or TRAF6. MiR-370-3p inhibition attenuated the protective effects of circ_0010729 knockdown on hypoxia-modulated cardiomyocyte dysfunction. MiR-370-3p restoration protected cardiomyocytes against hypoxic injury via targeting TRAF6. Besides, circ_0010729 indirectly regulated TRAF6 expression via miR-370-3p. This study demonstrated that circ_0010729 knockdown attenuated hypoxia-induced cardiomyocyte dysfunction via miR-370-3p/TRAF6 axis, indicating a potential therapeutic target for myocardial infarction.

Negative Effects of SIGIRR on TRAF6 Ubiquitination in Acute Lung Injury In Vitro.

In this study, the effects of single immunoglobin IL-1 receptor-related protein (SIGIRR) on tumor necrosis factor- (TNF-) receptor-associated factor 6 (TRAF6) ubiquitination in acute lung injury (ALI) were evaluated in both alveolar epithelial cells and alveolar macrophage cells in vitro. Our results found that SIGIRR negatively regulated TRAF6 ubiquitination and such SIGIRR inhibition could enhance the TRAF6 expression in both alveolar epithelial cells (AECs) and alveolar macrophage cells (AMCs). SIGIRR knockdown may increase NF-κB activity via TRAF6 regulation by the classical but not the nonclassical NF-κB signaling pathway. Such modulation between TRAF6 and SIGIRR could affect cytokine secretion and exacerbate the immune response; the IL-8, NFKB1, and NFKBIA mRNA levels were reduced after SIGIRR overexpression. The current study reveals the molecular mechanisms of the negative regulatory roles of SIGIRR on the innate immune response related to the LPS/TLR-4 signaling pathway and provides evidence for strategies to clinically treat inflammatory diseases.

꾸지뽕 열매 발효식초의 LPS에 의해 유도된 3T3-L1 세포 염증반응 억제 효과

Cudrania tricuspidata fruit has anti-inflammatory and anti-obesity effects. The present study aimed to determine the effect of C. tricuspidata fruits vinegar (CFV) on lipopolysaccharide (LPS)-induced adipocytes inflammation in 3T3-L1 adipocytes. The differentiation of 3T3-L1 adipocytes was induced by dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX) and insulin. Differentiated 3T3-L1 adipocytes were incubated with CFV followed by LPS treatment. The secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were measured using ELISA. The expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6) and nuclear factor kappa B (NF-κB) were measured by western blotting. The results showed that CFV dose-dependently inhibited lipid droplet generation and LPS-induced TNF-α, IL-6 and MCP-1 production in the adipocytes. CFV inhibited NF-κB activation, and the LPS-induced expression of TLR4, MyD88, and TRAF6. These results indicated that CFV inhibits the LPS-induced inflammatory response in adipocytes by inhibiting TLR4 signaling, including NF-κB pathways.

Temporal changes in inflammatory mitochondria-enriched microRNAs following traumatic brain injury and effects of miR-146a nanoparticle delivery.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and contribute to all aspects of cellular function. We previously reported that the activities of several mitochondria-enriched miRNAs regulating inflammation (i.e., miR-142-3p, miR-142-5p, and miR-146a) are altered in the hippocampus at 3–12 hours following a severe traumatic brain injury. In the present study, we investigated the temporal expression profile of these inflammatory miRNAs in mitochondria and cytosol fractions at more chronic post-injury times following severe controlled cortical impact injury in rats. In addition, several inflammatory genes were analyzed in the cytosol fractions. The analysis showed that while elevated levels were observed in cytoplasm, the mitochondria-enriched miRNAs, miR-142-3p and miR-142-5p continued to be significantly reduced in mitochondria from injured hippocampi for at least 3 days and returned to near normal levels at 7 days post-injury. Although not statistically significant, miR-146a also remained at reduced levels for up to 3 days following controlled cortical impact injury, and recovered by 7 days. In contrast, miRNAs that are not enriched in mitochondria, including miR-124a, miR-150, miR-19b, miR-155, and miR-223 were either increased or demonstrated no change in their levels in mitochondrial fractions for 7 days. The one exception was that miR-223 levels were reduced in mitochondria at 1 day following injury. No major alterations were observed in sham operated animals. This temporal pattern was unique to mitochondria-enriched miRNAs and correlated with injury-induced changes in mitochondrial bioenergetics as well as expression levels of several inflammatory markers. These observations suggested a potential compartmental re-distribution of the mitochondria-enriched inflammatory miRNAs and may reflect an intracellular mechanism by which specific miRNAs regulate injury-induced inflammatory signaling. To test this, we utilized a novel peptide-based nanoparticle strategy for in vitro and in vivo delivery of a miR-146a mimic as a potential therapeutic strategy for targeting nuclear factor-kappaB inflammatory modulators in the injured brain. Nanoparticle delivery of miR-146a to BV-2 or SH-SY5Y cells significantly reduced expression of TNF receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1), two important modulators of the nuclear factor-kappaB (NF-κB) pro-inflammatory pathway. Moreover, injections of miR-146a containing nanoparticles into the brain immediately following controlled cortical impact injury significantly reduced hippocampal TNF receptor-associated factor 6 and interleukin-1 receptor-associated kinase 1 levels. Taken together, our studies demonstrate the subcellular alteration of inflammatory miRNAs after traumatic brain injury and establish proof of principle that nanoparticle delivery of miR-146a has therapeutic potential for modulating pro-inflammatory effectors in the injured brain. All of the studies performed were approved by the University of Kentucky Institutional Animal Care and Usage Committee (IACUC protocol # 2014-1300) on August 17, 2017.

Cardioprotective role of GTS-21 by attenuating the TLR4/NF-κB pathway in streptozotocin-induced diabetic cardiomyopathy in rats.

The cholinergic anti-inflammatory pathway (CAP) was investigated in a variety of inflammatory conditions and constitutes a valuable line in their treatment. In the current study, we investigated the anti-inflammatory effect of GTS-21 (GTS) as a partial selective α7 nicotinic acetylcholine receptor (α7-nAchR) agonist in diabetic cardiomyopathy model in rats. This mechanism was elaborated to study whether it could alleviate the electrocardiographic, histopathological, and molecular levels of Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) pathway proteins. Diabetes was induced by the injection of streptozotocin (STZ) (50mg/kg). Diabetic rats were treated with GTS (1 or 2mg/kg/day), methyllycaconitine (MLA), a selective α7-nAchR antagonist (2mg/kg/day) plus GTS (2mg/kg/day), or the vehicle. All treatments were given by the intraperitoneal route. Ventricular rate and different electrocardiograph (ECG) anomalies were detected. Plasma levels of cardiac troponin T (cTnT) and creatine kinase MB (CK-MB) were measured by ELISA. Additionally, we elucidated the levels of several proteins involved in the TLR4/NF-κB pathway. Cardiac levels of TLR4 and phosphorylated protein kinase B (p-Akt) were detected by ELISA. The cardiac expression of myeloid differentiation primary response 88 (Myd88), tumor necrosis factor receptor-associated factor 6 (TRAF6), NF-κB, interleukin 1β (IL-1β), and active caspase-1 were evaluated by immunohistochemical staining. Finally, the cardiac levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by ELISA. Diabetic rats showed (i) ECG signs of cardiomyopathy such as significant ST segment elevations, prolonged QRS, QT intervals, and ventricular tachycardia; (ii) increased plasma levels of cTnT and CK-MB; (iii) increased expression of cardiac TLR4; (iv) elevated immunohistochemical expression of cardiac, Myd88, TRAF6, and NF-κB; (v) diminution in the cardiac expression of p-Akt; and (vi) adaptive increases in cardiac expression of TNF-α and IL-6. These effects were ameliorated in diabetic rats treated with both doses of GTS. Pretreatment with MLA did not completely reverse the ameliorative effect of GTS on cTnT, TRAF6, TNF-α, and IL-6, thereby reinforcing the presence of possible α7-nAchR-independent mechanisms. The activation of α7-nAchR with GTS offers a promising prophylactic strategy for diabetic cardiomyopathy by attenuating the TLR4/NF-κB pathway.

Long Non-Coding RNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)Relieves Sepsis-Induced Kidney Injury and Lipopolysaccharide (LPS)-Induced Inflammation in HK-2 Cells.

BackgroundLong non-coding RNAs (lncRNAs) play key roles in the development and progression of diseases, including sepsis. Therefore, this study aimed to clarify the role and underlying molecular mechanisms of lncRNA NEAT1 in sepsis.Material/MethodsWe used real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the expression of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), let-7b-5p, and tumor necrosis factor receptor-associated factor 6 (TRAF6). Western blot assay was used to measure the protein expression levels. After treatment with lipopolysaccharide (LPS), the biological behaviors of human renal tubular epithelial cells (HK-2), such as proliferation and apoptosis, were determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) and flow cytometry assays, respectively. The interaction relationship among NEAT1, TRAF6, and let-7b-5p was analyzed by the bioinformatics starBase database and dual-luciferase reporter assay.ResultslncRNA NEAT1 was expressed at higher levels in kidney tissues from sepsis patients than in healthy kidney tissues. Interestingly, LPS induced high expression of lncRNA NEAT1 in HK-2 cells in a time- and dose-dependent manner. Furthermore, silencing of NEAT1 weakened LPS-induced apoptosis, inflammation, and inhibition of proliferation, which was overturned by silencing of let-7b-5p. In addition, overexpression of TRAF6 abolished the overexpression of let-7b-5p-induced effects on apoptosis, inflammation, and growth of HK-2 cells exposed to LPS. In summary, NEAT1 regulated TRAF6 expression by sponging let-7b-5p in HK-2 cells, which promotes LPS-induced injury and inflammation in HK-2 cells.ConclusionsOur data show that the lower expression of NEAT1 impeded sepsis development and LPS-induced injury inflammation by targeting let-7b-5p/TRAF6 axis, and NEAT1 may be a target for treatment of sepsis patients.

By targeting TRAF6, miR-140-3p inhibits TGF-β1-induced human osteosarcomaepithelial-to-mesenchymal transition, migration, and invasion.

We evaluated the effects of miR-140-3p on EMT, cellular migration, and invasion in TGF-β1 treated human OS cells. Human fresh OS tissue and normal bone tissue specimens were gathered from 42 patients (29 male and 13 female, 11 to 24years of age with a mean age of 17.5 ± 2.3years) diagnosed with OS by pathology. By targeting TRAF6, miR-140-3p inhibits TGF-β1-induced human osteosarcoma epithelial-to-mesenchymal transition, migration, and invasion. In this study, we found microRNA (miR)-140-3p to be down-regulated and tumor necrosis factor receptor-associated factor 6 (TRAF6) to be up-regulated in patient OS samples. Lower levels of miR-140-3p and higher levels of TRAF6 were found in the advanced Enneking stage of OS. Furthermore, both mRNA and protein levels of TRAF6 were negatively associated with miR-140-3p mRNA expression in human OS tissue. TRAF6 was verified as a direct target of miR-140-3p in TGF-β1-treated human U2OS cells. Further, a miR-140-3p mimic dramatically inhibited while a miR-140-3p inhibitor enhanced TGF-β1-induced epithelial-to-mesenchymal transition, migration, and invasion of U2OS cells. Small interfering RNA was found to silence TRAF6 and to partly reverse the effects of the miR-140-3p inhibitor on TGF-β1-treated U2OS cells in vitro. These results demonstrate miR-140-3p to function as a tumor inhibitor of human OS cells by decreasing TRAF6 expression. miR-140-3p and TRAF6 may be valuable and novel biomarkers for diagnosis and treatment of OS.

Destablilization of TRAF6 by DRAK1 Suppresses Tumor Growth and Metastasis in Cervical Cancer Cells.

The adaptor protein TNF receptor-associated factor 6 (TRAF6) is a key mediator in inflammation. However, the molecular mechanisms controlling its activity and stability in cancer progression remain unclear. Here we show that death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) inhibits the proinflammatory signaling pathway by targeting TRAF6 for degradation, thereby suppressing inflammatory signaling-mediated tumor growth and metastasis in advanced cervical cancer cells. DRAK1 bound directly to the TRAF domain of TRAF6, preventing its autoubiquitination by interfering with homo-oligomerization, eventually leading to autophagy-mediated degradation of TRAF6. Depletion of DRAK1 in cervical cancer cells resulted in markedly increased levels of TRAF6 protein, promoting activation of the IL1β signaling-associated pathway and proinflammatory cytokine production. DRAK1 was specifically underexpressed in metastatic cervical cancers and inversely correlated with TRAF6 expression in mouse xenograft model tumor tissues and human cervical tumor tissues. Collectively, our findings highlight DRAK1 as a novel antagonist of inflammation targeting TRAF6 for degradation that limits inflammatory signaling-mediated progression of advanced cervical cancer. SIGNIFICANCE: Serine/threonine kinase DRAK1 serves a unique role as a novel negative regulator of the inflammatory signaling mediator TRAF6 in cervical cancer progression.

MicroRNA-4443 regulates monocyte activation by targeting tumor necrosis factor receptor associated factor 4 in stroke-induced immunosuppression.

MicroRNAs (miRNAs) have been demonstrated to play crucial roles in the early stage of acute ischaemic stroke (AIS). The purpose of this study was to investigate the expression patterns of miRNAs in peripheral blood mononuclear cells (PBMCs) from AIS patients and further explore related molecular mechanisms in stroke-induced immunodeficiency syndrome (SIDS). The miRNA expression patterns of PBMCs were detected by miRNA microarray and validated by quantitative real-time polymerase chain reaction (qRT-PCR) in AIS patients and healthy controls. Bioinformatics methods and luciferase reporter assays were used to detect the downstream target genes. Following stimulation with lipopolysaccharide and interleukin-4, the expression of miR-4443, tumor necrosis factor receptor associated factor 4 (TRAF4) and the nuclear factor kappa B (NF-κB) pathway were evaluated. Furthermore, transfection with miR-4443 mimic or inhibitor in the monocytes was carried out to gain insight into the mechanisms in SIDS. Interleukin-10 in AIS patients was significantly higher than that of healthy controls. The miRNA microarray analysis and qRTPCR validation showed that only miR-4443 was upregulated expressed in PBMCs from AIS patients, especially in monocytes. miR-4443 was shown to directly interact with the 3' untranslated regions of TRAF4, resulting in suppression of TRAF4 protein expression. Furthermore, the expression of miR-4443 and TRAF4 was regulated by stimulation with lipopolysaccharide or interleukin-4. Additionally, overexpression of miR-4443 suppressed the TRAF4/Iκα/NF-κB signaling pathway to activate the expression of anti-inflammatory cytokines in monocytes. The increased expression of miR-4443 induced monocyte dysfunction by targeting TRAF4, which may function as a crucial mediator in SIDS.

Molecular characterisation, expression and functional feature of TRAF6 in the King pigeon (Columba livia).

TNF receptor-associated factor 6 (TRAF6) is a signal transducer, which plays a pivotal role in triggering a variety of signalling cascades. Here, we cloned and identified the TRAF6 gene from the King pigeon. The open reading frame sequence of pigeon TRAF6 (piTRAF6) is 1638 bp long and encodes a 545 aa protein, including a low-complexity domain, RING finger, Zinc finger, coiled coil domain, and meprin and TRAF homology domain. The aa sequence of piTRAF6 shared a strong identity with that of other birds. PiTRAF6 transcripts were broadly expressed in all the tested tissues; piTRAF6 levels were the highest and lowest in the heart and stomach, respectively. Overexpression of piTRAF6 activated NF-κB in a dose-dependent manner and induced IFN-β expression. Upon piTRAF6 knockdown by small interfering RNAs, NF-κB activation was markedly inhibited in HEK293T cells. The expression of piTRAF6, as well as pro-inflammatory cytokines and antiviral molecules, were obviously increased after TLR ligand stimulation and Newcastle disease virus or Salmonella Pullorum inoculation. These results suggest that piTRAF6 may play a key immunoregulatory role in the innate immune response against viral and bacterial infections.

Effect and mechanism of miR-146a on malignant biological behaviors of lung adenocarcinoma cell line.

The aim of the present study was to assess the expression of microRNA-146a (miR-146a) in human lung adenocarcinoma cells, its effect on cellular behaviors, and the underlying molecular mechanisms. Reverse transcription-quantitative PCR (RT-qPCR) was used to measure miR-146a expression in the human normal lung epithelial cell line, BEAS-2B, and human lung adenocarcinoma cell lines, A549, PC-9 and H1299, to determine whether miR-146a acts as an oncogene or anti-oncogene. miR-146a mimics were transfected into target cells to observe the proliferation, apoptosis, invasion and migration of human lung adenocarcinoma cells. The target genes of miR-146a were predicted using bioinformatics analysis, and binding sites were validated by dual-luciferase reporter assay. Target gene expression at the mRNA and protein levels was measured by RT-qPCR and western blot analysis, respectively. The expression levels of miR-146a in human lung adenocarcinoma cell lines were lower than its expression in BEAS-2B (P<0.01). A549 cell line is a EGFR wild-type lung adenocarcinoma cell line, which is also the most widely studied in NSCLC, and therefore this was chosen as the target cell line for further investigation. Overexpression of miR-146a in A549 cells can inhibit cell proliferation (P<0.05), promote apoptosis (P<0.05), and reduce the cells' migratory ability (P<0.01). Bioinformatics prediction indicated that interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor 6 (TRAF6) are the target genes of miR-146a. Dual-luciferase reporter assay showed that miR-146a could specifically bind to 3′-untranslated regions of IRAK1 and TRAF6. The protein and mRNA levels of IRAK1 and TRAF6 were significantly downregulated after miR-146a overexpression in A549 cells (P<0.01). The results of this study demonstrated that the expression of miR-146a in human lung adenocarcinoma cells was significantly lower than in normal lung epithelial cells, indicating that miR-146a acts as an anti-oncogene. miR-146a suppresses the proliferation and migration of human lung adenocarcinoma cells by downregulating the expression of IRAK1 and TRAF6.

The Nephroprotective Effect of TNF Receptor-Associated Factor 6 (TRAF6) Blockade on LPS-Induced Acute Renal Injury Through the Inhibition if Inflammation and Oxidative Stress.

BackgroundInflammation and oxidative stress play important roles in the pathogenesis of acute kidney injury (AKI). TRAF6 functions as a signal transducer in the Toll-like receptor 4 signaling pathway. Several reports have previously implicated TRAF6 signaling in kidney pathology. Here, we investigated whether TRAF6 blockade can mitigate inflammatory responses and oxidative stress in AKI.Material/MethodsC57BL/6 mice were injected with lipopolysaccharide (LPS, 15 mg/kg) to induce AKI. Double immunofluorescence staining of kidney tissues showed that TRAF6 was localized to renal tubular epithelial cells, and then a tubular epithelial cell line (NRK-52E) was used for in vitro analysis. TRAF6 was blocked in vitro using siRNA and in vivo using AAV2/2 shRNA.ResultsThe knockdown of TRAF6 in mice by AAV2-shTRAF6 significantly reduced renal inflammation, oxidative stress, apoptosis and kidney dysfunction in AKI. In vitro, silencing the expression of TRAF6 attenuated LPS(0.5 μg/mL)-induced inflammatory responses and oxidative stress and upregulated proapoptotic factors. Furthermore, the beneficial actions of TRAF6 blockade were closely associated with its ability to increase IκB-α and Nrf2.ConclusionsOur findings provide direct evidence that TRAF6 mediates LPS-induced inflammation and oxidative stress, leading to renal dysfunction. We also show that TRAF6 inhibition is a potential therapeutic option to prevent AKI.

Impact of miR-SNP rs2910164 on miR-146a expression in osteoarthritic chondrocytes

PurposeMiR-146a acts as a negative inflammatory mediator in different diseases and has been implicated in osteoarthritis (OA) pathogenesis. In our study, we investigated the association between miR-SNP rs2910164 and OA susceptibility and its role on the expression of miR-146a, inflammatory and catabolic mediators in osteoarthritic chondrocytes. Materials and methodsGenetic association analysis was performed in 1688 knee OA patients and healthy individuals of Greek origin. Genomic DNA was extracted from blood and genotyped for rs2910164 (G > C) using Restriction-Fragment Length Polymorphism (RFLP). Total RNA was extracted from chondrocytes of 18 OA patients and miR-146a, IL-1 Receptor-Associated Kinase 1 (IRAK-1), TNF Receptor-Associated Factor 6 (TRAF-6), A Disintegrin and Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5), Matrix Metalloproteinase-13 (MMP-13), Interleukin-6 (IL-6), Interleukin-1 Beta (IL-1β) and Tumor Necrosis Factor-Alpha (TNF-α) expression was evaluated using quantitative Real-Time PCR (qRT-PCR). ResultsOA patients carrying rs2910164-GC and CC genotypes did not have an increased risk for OA development compared to GG genotype carriers. MiR-146a expression in OA chondrocytes was significantly lower in patients with rs2910164-GC genotype than in the GG carriers. OA patients carrying the rs2910164-GC genotype in their chondrocytes exhibited increased IRAK-1, TRAF-6, MMP-13, IL-1β and IL-6 expression levels compared with rs2910164-GG carriers. ConclusionWe demonstrate, for the first time, that miR-SNP rs2910164 in miR-146a gene is associated with reduced miR-146a and increased inflammatory and catabolic mediators’ expression in OA chondrocytes. Our data imply that genetic variations in miRNAs linked to OA pathogenesis may regulate their expression levels, suggesting new therapeutic strategies for patients with cartilage diseases.

Targeting spinal TRAF6 expression attenuates chronic visceral pain in adult rats with neonatal colonic inflammation.

BackgroundIrritable bowel syndrome is one of the most common gastrointestinal disorders. It is featured by abdominal pain in conjunction with altered bowel habits. However, the pathophysiology of the syndrome remains largely unknown. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been reported to be involved in neuropathic pain. The aim of this study was to investigate roles and mechanisms of TRAF6 in the chronic visceral hypersensitivity.MethodsVisceral hypersensitivity was induced by neonatal colonic inflammation and was identified by colorectal distention. The protein level, RNA level, and cellular distribution of TRAF6 and its related molecules were detected with Western blot, quantitative polymerase chain reaction, and immunofluorescence. In vitro spinal cord slice recording technique was performed to determine the synaptic transmission activities.ResultsNeonatal colonic inflammation rats displayed visceral hypersensitivity at the age of six weeks. The expression of TRAF6 was obviously upregulated in spinal cord dorsal horn of neonatal colonic inflammation rats at the age of six weeks. Immunofluorescence study showed that TRAF6 was dominantly expressed in spinal astrocytes. Intrathecal injection of TRAF6 small interfering RNA (siRNA) significantly reduced the amplitude of spontaneous excitatory postsynaptic currents at the spinal dorsal horn level. Furthermore, knockdown of TRAF6 led to a significant downregulation of cystathionine β synthetase expression in the spinal dorsal horn of neonatal colonic inflammation rats. Importantly, intrathecal injection of TRAF6 siRNA remarkably alleviated visceral hypersensitivity of neonatal colonic inflammation rats.ConclusionsOur results suggested that the upregulation of TRAF6 contributed to visceral pain hypersensitivity, which is likely mediated by regulating cystathionine β synthetase expression in the spinal dorsal horn. Our findings suggest that TRAF6 might act as a potential target for the treatment of chronic visceral pain in irritable bowel syndrome patients.

BAFF is involved in the pathogenesis of IgA nephropathy by activating the TRAF6/NF‑κB signaling pathway in glomerular mesangial cells.

The aim of the present study was to investigate the involvement of B cell-activating factor (BAFF) in the pathogenesis of IgA nephropathy by activating the tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB signaling pathway in glomerular mesangial cells. For the clinical analysis, blood, urine and kidney tissue samples were collected from 58 patients diagnosed with primary IgA nephropathy by renal biopsy. For the in vitro study, glomerular mesangial cells were divided into five groups: Control (con)-short hairpin RNA (shRNA) (control group); con-shRNA + BAFF (20 ng/ml); con-shRNA + BAFF + BAFF-RFc chimera protein (500 µg/ml); TRAF6-shRNA; and TRAF6-shRNA + BAFF (20 ng/ml). For the in vivo experiments, 60 Sprague-Dawley rats were randomly divided into four groups: Con-small interfering RNA (siRNA) (control group); con-siRNA + IgA (IgA nephropathy group), BAFF-RFc chimera protein (2 µg/ml) + IgA, and TRAF6-siRNA (0.2 µM) + IgA. Reverse transcription-quantitative PCR was performed to evaluate the mRNA expression levels of TRAF6, connective tissue growth factor (CTGF), fibronectin (FN) and NF-κBP65. Western blot analysis was used to detect the protein expression levels of TRAF6, FN, CTGF and phosphorylated-NF-κBP65 in glomerular mesangial cells and kidney tissues. The results revealed that plasma BAFF levels were positively correlated with the severity of pathological damage in patients with IgA nephropathy. In vitro, BAFF induced the mRNA and protein expression of TRAF6, CTGF, FN and NF-κBP65 in glomerular mesangial cells. After the BAFF-RFc chimera protein was added to inhibit the binding of BAFF and BAFF-receptor (-R), this effect was reduced. In vivo, inhibition of the effects of BAFF via injection with the BAFF-R Fc chimera protein reduced kidney damage in rats suffering from IgA nephropathy. The effect on the expression of signaling pathway-associated proteins was also alleviated. In conclusion, BAFF enhanced the expression of fibroblast factors in the kidneys by activating the TRAF6/NF-κB signaling pathway.

A hMTR4-PDIA3P1-miR-125/124-TRAF6 Regulatory Axis and Its Function in NF kappa B Signaling and Chemoresistance.

Background and AimsDNA damage‐induced NF‐κB activation is a major obstacle to effective antitumour chemotherapy. Long noncoding RNAs (lncRNAs) that regulate chemoresistance of cancer cells remain largely unknown. This study aimed to characterize the lncRNAs that may affect chemotherapy sensitivity.Approach and ResultsWe found that lncRNA PDIA3P1 (protein disulfide isomerase family A member 3 pseudogene 1) was up‐regulated in multiple cancer types and following treatment with DNA‐damaging chemotherapeutic agents, like doxorubicin (Dox). Higher PDIA3P1 level was associated with poorer recurrence‐free survival of human hepatocellular carcinoma (HCC). Both gain‐of‐function and loss‐of‐function studies revealed that PDIA3P1 protected cancer cells from Dox‐induced apoptosis and allowed tumor xenografts to grow faster and to be more resistant to Dox treatment. Mechanistically, miR‐125a/b and miR‐124 suppressed the expression of tumor necrosis factor receptor‐associated factor 6 (TRAF6), but PDIA3P1 bound to miR‐125a/b/miR‐124 and relieved their repression on TRAF6, leading to activation of the nuclear factor kappa B (NF‐κB) pathway. Consistently, the effect of PDIA3P1 inhibition in promoting Dox‐triggered apoptosis was antagonized by silencing the inhibitor of κBα (IκBα) or overexpressing TRAF6. Administration of BAY 11‐7085, an NF‐κB inhibitor attenuated PDIA3P1‐induced resistance to Dox treatment in mouse xenografts. Moreover, up‐regulation of PDIA3P1 was significantly correlated with elevation of TRAF6, phosphorylated p65, or NF‐κB downstream anti‐apoptosis genes in human HCC tissues. These data indicate that enhanced PDIA3P1 expression may confer chemoresistance by acting as a microRNA sponge to increase TRAF6 expression and augment NF‐κB signaling. Subsequent investigations into the mechanisms of PDIA3P1 up‐regulation revealed that human homologue of mRNA transport mutant 4 (hMTR4), which promotes RNA degradation, could bind to PDIA3P1, and this interaction was disrupted by Dox treatment. Overexpression of hMTR4 attenuated Dox‐induced elevation of PDIA3P1, whereas silencing hMTR4 increased PDIA3P1 level, suggesting that Dox may up‐regulate PDIA3P1 by abrogating the hMTR4‐mediated PDIA3P1 degradation.ConclusionThere exists a hMTR4‐PDIA3P1‐miR‐125/124‐TRAF6 regulatory axis that regulates NF‐κB signaling and chemoresistance, which may be exploited for anticancer therapy.

Resveratrol protects BV2 mouse microglial cells against LPS-induced inflammatory injury by altering the miR-146a-5p/TRAF6/NF-κB axis

Objective: To investigate the role of miR-146a-5p in the effects of resveratrol (RSV) on inflammatory response in BV2 mouse microglial cells.Materials and methods: BV2 cells were pretreated by RSV and stimulated with lipopolysaccharide (LPS). Cell Viability was checked using a MTT assay. Real-Time PCR was performed to detect the levels of pro-inflammatory cytokines (tumor necrosisfactor-α－TNF-α, interleukin-1β－IL-1β and interleukin-6 － IL-6) and miR-146a-5p expression. Western blot was used to analyze the protein expression of TNF receptor associated factor 6 (TRAF6) and phospho-nuclear factor kappa B (pNF-κB). Gain-of-function and loss-of-function analysis of miR-146a-5p was performed using transfection of miR-146a-5p mimic and miR-146a-5p inhibitor, respectively.Results: Pretreatment with RSV significantly and dose dependently inhibited LPS-induced production of TNF-α, IL-1β and IL-6 in BV2 cells. MiR-146a-5p was significantly upregulated after LPS treatment, and further increased in RSV and LPS-co-treated cells. MiR-146a-5p overexpression via miR-146a-5p mimic transfection downregulated the mRNA level of TNF-α, IL-1β and IL-6, as well as abrogated the protein expression of TRAF6 and pNF-κB in BV2 cells exposed to LPS. More importantly, the reducion of TNF-α, IL-1β and IL-6 level by RSV were reversed by miR-146a-5p silence via miR-146a-5p inhibitor transfection. Furthermore, silencing miR-146a-5p attenuated the inhibitory effect of RSV on the TRAF6/NF-κB pathway which was activated after induction with LPS. Conclusions: RSV can suppress LPS-induced inflammatory injury via modulating the miR-146a-5p/TRAF6/NF-κB axis in BV2 mouse microglial cells.

Tumor necrosis factor receptor associated factor 4 promotes invasion and metastasis of colorectal cancer cell line LoVo

Objective To study the effect of low expression of tumor necrosis factor receptor associated factor 4 (TRAF4) on invasion and migration of colorectal cancer cells. Methods Immunohistochemistry was used to detect the expression of TRAF4 in clinical specimens of colorectal cancer and the adjacent tissues. The expression levels of epithelial-mesenchymal transition (EMT)-related proteins [E-cadherin (E-cad), Snail and Slug] were detected by Western blotting. The Transwell assay was used to examine the invasive ability and migration ability of tumor cells LoVo after silencing TRAF4. Results Immunohistochemistry showed that TRAF4 expression was significantly higher in cancer tissues than in adjacent tissues (t=8.322, P<0.01). After transfection with TRAF4 small interfering RNA (siRNA), the expression of TRAF4 was significantly decreased (t=7.687, P<0.01), the expression of E-cad was increased (t=4.322, P<0.05), the expression of Snail and Slug was decreased (t=9.831, P<0.01, t=8.677, P<0.01), and the invasion and migration ability was obviously decreased (t=9.553, P<0.01, t=11.290, P<0.01). Conclusion Silencing TRAF4 can inhibit the invasion and migration of colorectal cancer cell LoVo. Key words: Colorectal cancer; Tumor necrosis factor receptor associated factor 4; Invasion; Migration; Epithelial-mesenchymal transition

Tumor Necrosis Factor Receptor-Associated Factor 6 Promotes Hepatocarcinogenesis by Interacting With Histone Deacetylase 3 to Enhance c-Myc Gene Expression and Protein Stability.

The oncogene c-Myc is aberrantly expressed and plays a key role in malignant transformation and progression of hepatocellular carcinoma (HCC). Here, we report that c-Myc is significantly up-regulated by tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, in hepatocarcinogenesis. High TRAF6 expression in clinical HCC samples correlates with poor prognosis, and the loss of one copy of the Traf6 gene in Traf6+/- mice significantly impairs liver tumorigenesis. Mechanistically, TRAF6 first interacts with and ubiquitinates histone deacetylase 3 (HDAC3) with K63-linked ubiquitin chains, which leads to the dissociation of HDAC3 from the c-Myc promoter and subsequent acetylation of histone H3 at K9, thereby epigenetically enhancing the mRNA expression of c-Myc. Second, the K63-linked ubiquitination of HDAC3 impairs the HDAC3 interaction with c-Myc and promotes c-Myc protein acetylation, which thereby enhances c-Myc protein stability by inhibiting carboxyl terminus of heat shock cognate 70-kDa-interacting protein-mediated c-Myc ubiquitination and degradation. Importantly, TRAF6/HDAC3/c-Myc signaling is also primed in hepatitis B virus-transgenic mice, unveiling a critical role for a mechanism in inflammation-cancer transition. In clinical specimens, TRAF6 positively correlates with c-Myc at both the mRNA and protein levels, and high TRAF6 and c-Myc expression is associated with an unfavorable prognosis, suggesting that TRAF6 collaborates with c-Myc to promote human hepatocarcinogenesis. Consistently, curbing c-Myc expression by inhibition of TRAF6 activity with a TRAF6 inhibitor peptide or the silencing of c-Myc by small interfering RNA significantly suppressed tumor growth in mice. Conclusion: These findings demonstrate the oncogenic potential of TRAF6 during hepatocarcinogenesis by modulating TRAF6/HDAC3/c-Myc signaling, with potential implications for HCC therapy.

MicroRNA-124 regulates TRAF6 expression and functions as an independent prognostic factor in colorectal cancer.

An increasing number of studies have confirmed that miR-124 exhibits a suppressive role in glioblastoma, cervical cancer and breast cancer; however, the function of miR-124 in colorectal cancer (CRC) has not been completely elucidated. In the present study, miR-124 expression was confirmed by reverse transcription-quantitative PCR in 80 colorectal tissues and para-cancerous tissues. The influence of altered miR-124 expression was analyzed by statistical approaches including Cox multivariate regression analysis and the Kaplan-Meier method, and the target genes of miR-124 were confirmed by luciferase reporter assays. Immunohistochemical techniques were also performed in order to measure the expression levels of target proteins. miR-124 expression was observed to be decreased in colorectal tissue samples, and this phenomenon was correlated with adverse clinical indicators and poor patient survival time. Luciferase reporter assays indicated that miR-124 directly regulated TNF receptor associated factor 6 (TRAF6) 3′-untranslated region (UTR). Hence, it was proposed that miR-124 dysregulation may negatively influence the expression of TRAF6 and therefore serve as a biomarker of epithelial-mesenchymal transition in CRC tissues. In summary, the present study demonstrated that miR-124 regulates the expression of TRAF6, and may potentially function as an independent prognostic factor and therapeutic target in patients with CRC.