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Increasing evidence reveals that physical exercise is an efficient therapeutical approach in the treatment of insulin resistance (IR) and related metabolic diseases. However, the potential beneficial effects of exercise on insulin resistance and its underlying mechanisms remain unclear. Recent findings elucidated the negative role of ASK1 in repressing the glucose uptake through JNK1‐IRS1‐Akt signalling in liver. Thus, a detailed investigation of the effect of ASK1‐mediated insulin signalling on exercise‐mediated improvement of insulin sensitivity and its underlying mechanism was implemented in this study. Using a high‐fat diet‐induced IR rat model of chronic or acute swimming exercise training, we here showed that body weight and visceral fat mass were significantly reduced after chronic exercise. Moreover, chronic exercise reduced serum FFAs levels and hepatic triglyceride content. Both chronic and acute exercise promoted glucose tolerance and insulin sensitivity. Meanwhile, both chronic and acute exercise decreased ASK1 phosphorylation and improved JNK1‐IRS1‐Akt signalling. Furthermore, exercise training decreased CFLAR, CREG and TRAF1 protein levels in liver of obese rats, which are positive regulator of ASK1 activity. These results suggested that swimming exercise demonstrated to be an effective ameliorator of IR through the regulation of ASK1‐mediated insulin signalling and therefore, could present a prospective therapeutic mean towards the treatment of IR and several metabolic diseases based on IR, containing NAFLD and type Ⅱ diabetes.

Background Angiogenesis plays an important role in the occurrence, development and metastasis of hepatocellular carcinoma (HCC). According to previous studies, miR-378a participates in tumorigenesis and tumor metastasis, but its exact role in HCC angiogenesis remains poorly understood. Methods qRT-PCR was used to investigate the expression of miR-378a-3p in HCC tissues and cell lines. The effects of miR-378a-3p on HCC in vitro and in vivo were examined by Cell Counting Kit-8 (CCK-8), Transwell, tube formation and Matrigel plug assays, RNA sequencing, bioinformatics, luciferase reporter, immunofluorescence and chromatin immunoprecipitation (ChIP) assays were used to detect the molecular mechanism by which miR-378a-3p inhibits angiogenesis. Results We confirmed that miR-378a-3p expression was significantly downregulated and associated with higher microvascular density (MVD) in HCC; miR-378a-3p downregulation indicated a short survival time in HCC patients. miR-378a-3p knockdown led to a significant increase in angiogenesis in vitro and in vivo. We found that miR-378a-3p directly targeted TNF receptor associated factor 1 (TRAF1) to attenuate NF-κB signaling, and then downregulated secreted vascular endothelial growth factor. DNA methyltransferase 1 (DNMT1)-mediated hypermethylation of miR-378a-3p was responsible for downregulating miR-378a-3p. Moreover, a series of investigations indicated that p65 initiated a positive feedback loop that could upregulate DNMT1 to promote hypermethylation of the miR-378a-3p promoter. Conclusion Our study indicates a novel DNMT1/miR-378a-3p/TRAF1/NF-κB positive feedback loop in HCC cells, which may become a potential therapeutic target for HCC.

Background and objective: This study aimed to explore the expression of TRAF1 in vitro kidney injury model, and the function mechanism of TRAF1 in the model growth and apoptosis. Methods: After transfecting HK2 cells with short hair RNA (shRNA), shTRAF1 gene silencing model was established. The cells were divided into shRNA group and shNC group. For kidney injury model, we used hypoxia/reoxygenation to establish H/R cell lines. MTT assay was used to determine cell viability. PI/FITC staining was used to determine cell apoptosis. The genes expressions were determined by RT-qPCR and western blotting, respectively. The concentration of MDA, SOD, iNOS and LDH was determined by ELISA. Results: The results of RT-qPCR and western blotting assay revealed that TRAF1 upregulated expression in AKI model cells. The results of MTT assay revealed that shRNA group exhibited significantly higher cell viability and lower cell apoptosis compared with the control group in H/R HK2 cells. In addition, TRAF1 downregulated expression inhibits oxidative stress response in H/R treated HK2 cell. Mechanically, TRAF1 deficiency protects HK2 cell via inhibiting p38-MAPK pathway. Conclusions: Our study suggests that TRAF1 could be a target in kidney injury treatment.

Preeclampsia is believed to be caused by impaired placentation with insufficient trophoblast invasion, leading to impaired uterine spiral artery remodeling and angiogenesis. However, the underlying molecular mechanism remains unknown. We recently carried out transcriptome profiling of placental long noncoding RNAs (lncRNAs) and identified 383 differentially expressed lncRNAs in early-onset severe preeclampsia. Here, we are reporting our identification of lncRNA INHBA-AS1 as a potential causal factor of preeclampsia and its downstream pathways that may be involved in placentation. We found that INHBA-AS1 was upregulated in patients and positively correlated with clinical severity. We systematically searched for potential INHBA-AS1-binding transcription factors and their targets in databases and found that the targets were enriched with differentially expressed genes in the placentae of patients. We further demonstrated that the lncRNA INHBA-AS1 inhibited the invasion and migration of trophoblast cells through restraining the transcription factor CENPB from binding to the promoter of TNF receptor-associated factor 1 (TRAF1). Therefore, we have identified the dysregulated pathway “INHBA-AS1-CENPB-TRAF1” as a contributor to the pathogenesis of preeclampsia through prohibiting the proliferation, invasion, and migration of trophoblasts during placentation., Graphical Abstract, Preeclampsia is believed to be caused by impaired placentation. Xinping Yang and colleagues report the identification of lncRNA INHBA-AS1 as a potential causal factor of preeclampsia. They found that INHBA-AS1 is positively correlated with clinical severity and involved in the pathogenesis through inhibiting the invasion and migration of trophoblast cells.

Aim/Objective: Ulcerative colitis (UC) is a chronic inflammatory bowel disease. In UC, a wide range of criteria are used for disease remission, with few studies investigating the differences between disease remission and normal control groups. This paper compares known inflammatory and healing mediators in the mucosa of UC in clinical remission and normal controls, in order to better describe the remission state. Method: Mucosal biopsies from 72 study participants (48 UC and 24 normal controls) were included from the Advanced Study of Inflammatory Bowel Disease (ASIB Study), Arctic University of Norway, Norway. Clinical remission was defined as Mayo clinical score ≤ 2, with endoscopic subscores of ≤ 1. Targeted gene transcription analyses were performed using hydrolysis probes and SYBR-green. Results: Among the mucosal transcripts examined, 10 genes were regulated in remission versus normal controls, 8 upregulated pro-inflammatory transcripts (IL1B, IL33, TNF, TRAF1, CLDN2, STAT1, STAT3 and IL13Ra2) and 2 downregulated (pro-inflammatory TBX21 and anti-inflammatory TGFB1). In total, 14 transcripts were regulated between the investigated groups. Several master transcription factors for T-cell development were upregulated in patients with Mayo endoscopic score of 1 in comparison to 0. Conclusions: The mucosa of UC in clinical and endoscopic remission differs from normal mucosa, suggesting a remaining dysregulation of inflammatory and wound healing mechanisms.

The prime issue derived from prostate cancer (PCa) is its high prevalence to metastasize to bone. MicroRNA-204-5p (miR-204-5p) has been reported to be involved in the development and metastasis in a variety of cancers. However, the clinical significance and biological functions of miR-204-5p in bone metastasis of PCa are still not reported yet. In this study, we find that miR-204-5p expression is reduced in PCa tissues and serum sample with bone metastasis compared with that in PCa tissues and serum sample without bone metastasis, which is associated with advanced clinicopathological characteristics and poor bone metastasis-free survival in PCa patients. Moreover, upregulation of miR-204-5p inhibits the migration and invasion of PCa cells in vitro, and importantly, upregulating miR-204-5p represses bone metastasis of PCa cells in vivo. Our results further demonstrated that miR-204-5p suppresses invasion, migration, and bone metastasis of PCa cells via inactivating nuclear factor κB (NF-κB) signaling by simultaneously targeting TRAF1, TAB3, and MAP3K3. In clinical PCa samples, miR-204-5p expression negatively correlates with TRAF1, TAB3, and MAP3K3 expression and NF-κB signaling activity. Therefore, our findings reveal a new mechanism underpinning the bone metastasis of PCa, as well as provide evidence that miR-204-5p might serve as a novel serum biomarker in bone metastasis of PCa. This study identifies a novel functional role of miR-204-5p in bone metastasis of prostate cancer and supports the potential clinical value of miR-204-5p as a serum biomarker in bone metastasis of PCa.

Background: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) achieves remarkable success in the treatment of patients with advanced non-small cell lung cancer (NSCLC) bearing activating EGFR mutations. However, EGFR-TKI resistance is still a obstacles in the clinical practice. TRAF1 as a member of TRAF family participates NSCLC progression. In our previous research found that TRAF1 played an important role in the progression of NSCLC.Methods: In vivo and vitro, quantitative PCR (qPCR) and western blot (WB) were carried out to check the expression of TRAF1 in gefitinib-sensitive and -resistant NSCLC samples. The cell counting kit-8 (CCK8) and flow cytometry were used to measure cell proliferation, apoptosis and cycle arrest.Results: Here we showed that TRAF1 over-expression is associated with clinical resistance to EGFR TKI. TRAF1 was significantly up-regulated in gefitinib-resistant cells. Knockdown of TRAF1 increased the sensitivity of gefitinib-resistant cell. Knockdown of TRAF1 induced cell apoptosis and cell cycle arrest. Conclusions: Therefore, our results highlight the function of TRAF1 in the EGFT-TKI resistance.

The present study was conducted in order to study the detailed molecular mechanism of tumor necrosis factor (TNF)-α in chronic obstructive pulmonary disease (COPD). The rats were treated with cigarette smoke (CS) and lipopolysaccharide (LPS) to establish the COPD model. Next, the changes in lung injury in COPD rats with TNF-α knockdown was tested. Meanwhile, the regulation of TNF-α on MAPK pathway and its downstream molecules (SOCS3/TRAF1) was determined by western blotting. On this basis, the activation of MAPK and inhibition of SOCS3/TRAF1 was also examined. Subsequently, the lung function was tested with the plethysmograph, the cells of bronchoalveolar lavage fluid was counted and classified. Furthermore, lung tissue sections were stained with hematoxylin and eosin to verify whether the treatment of MAPK pathway and downstream molecules affected the effect of TNF-α knockdown on COPD. The present study showed that TNF-α knockdown could alleviate the decrease in the function and inflammatory injury of the lungs of rats with COPD. Western blot analysis verified that TNF-α knockdown could inhibit the activation of MAPK pathway and increase the expression of SOCS3/TRAF1. The following experimental results showed that the relief of lung injury caused by TNF-α knockdown could be deteriorated by activating MAPK pathway. It was also found that the symptom of COPD was decreased following transfection with sh-TNF-α but worsened by SOCS3/TRAF1 knockdown. Overall, TNF-α knockdown inhibited the activation of MAPK pathway and increased the expression of SOCS3/TRAF1, thus delaying the process of COPD.

Made available in DSpace on 2020-12-10T19:47:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with the presence of chemoresistance contributing to the poor prognosis. Heat Shock Proteins (HSPs) genes are activated in response to pathophysiological stress and serve a role in a variety of stages in carcinogenesis, acting primarily as anti-apoptotic agents and in chemotherapy resistance in a variety of tumor types. The current study evaluated the HSP gene expression profile in women with ovarian cancer (OC) and their correlation with clinical and pathological aspects of patients with OC. A total of 51 patients included in the current study were divided into four groups: Primary Epithelial Ovarian Cancer (EOC; n=14), metastatic EOC (n=11), ovarian serous cystadenoma (n=7) and no evidence of ovarian malignancy or control groups (n=19). RNA extraction and reverse transcription-quantitative (RT-q) PCR was then performed on the samples obtained. RT-qPCR was performed to compare TNF receptor associated protein 1 (TRAP]), heat shock protein family (HSP) HSPB1, HSPD1, HSPA1A and HSPA1L expression in primary and metastatic EOCs. TRAP], HSPB1, HSPD1, HSPA1A and HSPAlL gene expression did not differ among groups. HSPA1A, HSPA]L and TRAP] were revealed to be underexpressed in the primary and metastatic EOC groups, with HSPA1L exhibiting the lowest expression. TRAP] expression was higher in tumors at stages I/II compared with those at stages III/IV. No correlation was exhibited between HSP expression and age, menarche, menopause, parity, period after menopause initiation, cytoreduction, CA-125 or overall and disease-free survival. HSPA1A was negatively correlated with the risk of mortality from OC. The results indicated that the downregulation of HSPA1A, HSPA1L and TRAP] could be associated with the clinical prognostic features of women with EOC. Vera Cruz Hosp, Oncol Serv, BR-30180090 Belo Horizonte, MG, Brazil Sao Paulo State Univ, Sch Med, Dept Obstet & Gynecol, BR-18618687 Botucatu, SP, Brazil Ezequiel Dias Fdn, Res & Dev Dept, Cellular Biol Serv, BR-30510010 Belo Horizonte, MG, Brazil Minas Gerais Fed Univ, Sch Med, Dept Obstet & Gynecol, 190 Alfredo Balena Ave, BR-30130100 Belo Horizonte, MG, Brazil Sao Paulo State Univ, Sch Med, Dept Obstet & Gynecol, BR-18618687 Botucatu, SP, Brazil FAPEMIG: CDS-APQ-02373-17

Long noncoding RNAs (lncRNAs), once thought to be transcriptional noise, have been recently shown to regulate a variety of biological processes. However, their roles in the inflammatory response are largely unexplored. In this study, we performed high-throughput sequencing to identify the profiles of mRNA and lncRNA transcriptomes in response to lipopolysaccharide (LPS) stimulation, followed by a comprehensive bioinformatics analysis. We found a total of 325 lncRNAs and 1,187 mRNAs to be significantly dysregulated in RAW264.7 cells stimulated with LPS (fold change >4.0 or

The results on associations of tumor necrosis factor (TNF)-receptor associated factor 1/complement component 5 (TRAF1/C5) rs10818488 and rs3761847 polymorphisms with rheumatoid arthritis (RA) are controversial, thus this study was performed to examine whether the aforementioned polymorphisms were associated with RA in a Chinese population. Furthermore, an updated meta-analysis was conducted. The polymorphisms were genotyped in 328 Chinese RA patients and 449 healthy controls. Studies examining the association of TRAF1/C5 rs10818488 and/or rs3761847 polymorphism with RA were exhaustively searched. No significant difference in either genotype or allele distribution between RA patients and controls was found. The updated meta-analysis was conducted based on 19 articles including the present study. A significant association of RA with TRAF1/C5 rs10818488 polymorphism G allele in Europeans (OR = 0.843, 95% CI = 0.730-0.975, p = 0.021) and in Asians (OR = 1.070, 95% CI = 1.009-1.136, p = 0.024) was found. Additionally, a significant association of RA with TRAF1/C5 rs10818488 polymorphism G allele under the recessive model in Asians (OR = 1.129, 95% CI = 1.023-1.246, p = 0.016) and in Africans (OR = 0.657, 95% CI = 0.507-0.851, p = 0.001) was found. Only a borderline significant association of RA with TRAF1/C5 rs3761847 polymorphism A allele was found in Europeans. Non-significant associations of RA with TRAF1/C5 rs10818488 and rs3761847 polymorphisms were found in our study. The updated meta-analysis results demonstrate that TRAF1/C5 rs10818488 polymorphism is associated with RA in Europeans, Asians and Africans, and TRAF1/C5 rs3761847 polymorphism is associated with RA in Europeans with borderline significant evidence.

TRAF1 is a pro-survival adaptor molecule in TNFR superfamily (TNFRSF) signaling. TRAF1 is overexpressed in many B cell cancers including refractory chronic lymphocytic leukemia (CLL). Little has been done to assess the role of TRAF1 in human cancer. Here we show that the protein kinase C related kinase Protein Kinase N1 (PKN1) is required to protect TRAF1 from cIAP-mediated degradation during constitutive CD40 signaling in lymphoma. We show that the active phospho-Thr774 form of PKN1 is constitutively expressed in CLL but minimally detected in unstimulated healthy donor B cells. Through a screen of 700 kinase inhibitors, we identified two inhibitors, OTSSP167, and XL-228, that inhibited PKN1 in the nanomolar range and induced dose-dependent loss of TRAF1 in RAJI cells. OTSSP167 or XL-228 treatment of primary patient CLL samples led to a reduction in TRAF1, pNF-κB p65, pS6, pERK, Mcl-1 and Bcl-2 proteins, and induction of activated caspase-3. OTSSP167 synergized with venetoclax in inducing CLL death, correlating with loss of TRAF1, Mcl-1, and Bcl-2. Although correlative, these findings suggest the PKN1-TRAF1 signaling axis as a potential new target for CLL. These findings also suggest the use of the orally available inhibitor OTSSP167 in combination treatment with venetoclax for TRAF1 overexpressing CLL.

Pneumonia is a persistent and pervasive disease, the effects of which can be severe. MicroRNA (miR)-127-5p has been utilized as a novel biomarker for the diagnosis of severe pneumonia. The present study aimed to investigate the function of miR-127-5p during severe pneumonia. An in vitro model of severe pneumonia in Ana-1 murine macrophages was established using lipopolysaccharide (LPS). Subsequently, reverse transcription-quantitative PCR and ELISA were performed to detect the mRNA and protein expression levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. Western blotting was also performed to measure the activity of AKT and NF-κB. The results indicated that compared with the control group, LPS treatment increased TNF receptor-associated factor 1 (TRAF1) expression levels and reduced miR-127-5p expression levels. Furthermore, the results revealed that the 3'-untranslated region of TRAF1 was targeted by miR-127-5p. miR-127-5p mimic reduced LPS-induced increases in IL-1β, IL-6 and TNF-α expression by targeting TRAF1, which was potentially mediated by inactivation of the AKT and NF-κB signaling pathways. Collectively, the results demonstrated that miR-127-5p may attenuate severe pneumonia by reducing LPS-induced inflammatory cytokine production, and inactivating the AKT and NF-κB signaling pathways by targeting TRAF1.

Objective: The purpose of this study was to explore the effect of TRAF1 on phenotypic changes of KCs in I/R in liver transplantation. Methods: SD rats were randomly divided into sham group and liver transplantation I/R group. KCs were extracted from rat livers in each group. KCs were transfected by lentivirus of si-TRAF1 or si-HOIP, and induced by Lipopolysaccharide (LPS). Flow cytometry was used to detect cell apoptosis. Western blot and RT-PCR were used to detect the protein and mRNA expression levels. Results: Compared with the sham group, the expression levels of TRAF1, TNF-α and IL-1β were significantly increased in the I/R group (P Conclusion: TRAF1 was an important negative regulator of liver transplantation I/R by inhibiting the activation of NF-κB in KCs and preventing its M1 phenotype transformation.

TNF Receptor Associated Protein 1 (TRAP1) is a mitochondrial paralog of Hsp90 related to the promotion of tumorigenesis in various cancers via maintaining mitochondrial integrity, reducing the production of reactive oxygen species, and reprogramming cellular metabolism. Consequently, Hsp90 and TRAP1 have been targeted to develop cancer therapeutics. Herein, we report a series of pyrazolo[3,4-d]pyrimidine derivatives that are mitochondria-permeable TRAP1 inhibitors. Structure-based drug design guided the optimization of potency, leading to the identification of compounds 47 and 48 as potent TRAP1 and Hsp90 inhibitors with good metabolic and plasma stability as well as acceptable CYP and hERG inhibition. X-ray co-crystallization studies confirmed both 47 and 48 interact with the ATP binding pocket in the TRAP1 protein. Compounds 47 and 48 demonstrated excellent anticancer efficiency in various cancer cells, with limited toxicity over normal hepatocyte and prostate cells. Mouse PC3 xenograft studies showed 47 and 48 significantly reduced tumor growth.

Objective. To investigate the effects of HuR protein on the treatment of chronic lymphocytic leukemia (CLL). Methods. LCL lymphoblast cells and B lymphocytes were subjected to HuR overexpression (OV) or interference (IV). Western blot was used to observe the protein expression of human tumor necrosis factor-associated factor 1 (TRAF1), human inhibitor of nuclear factor kappa-B kinase α (IKK-α), NF-κB-inducing kinase (NIK), and p52. Flow cytometry was performed to evaluate apoptosis, and the mRNA expression of TRAF1 was examined by quantitative reverse transcription polymerase chain reaction. Immunofluorescence was carried out to visualize the expression of HuR, and the relationship between HuR and TRAF1 was observed by pull-down test. Cell sensitivity to chlorambucil (CLB) and fludarabine (Flu) was assessed by Cell Counting Kit-8. Results. The expression of HuR and TRAF1 in LCLs was significantly increased compared to that in B lymphocytes. Compared with the control, HuR OV significantly increased the expression of TRAF1 (P<0.05), whereas it was significantly decreased in the IV group (P<0.05). HuR can bind to TRAF1 directly, and the binding rate is positively correlated with HuR expression. After inhibiting HuR, the expression of TRAF1, IKK-α, NIK, p52, pro-Caspase 3, and PARP was significantly upregulated in LCLs and B lymphocytes (P<0.05), while Caspase 3 was downregulated (P<0.05). Compared with the control, the proliferation of LCLs and B lymphocytes treated by CLB and Flu decreased significantly after HuR blockade (P<0.05). Conclusion. HuR may be a key protein regulating CLL resistance. After inhibiting HuR, inflammatory response and apoptosis were significantly increased, and the cell sensitivity to CLB and Flu increased, suggesting that inhibiting HuR activity may be a potential strategy to solve the problem of drug resistance in CLL cells.

Genetic studies have implicated both CD40 and tumour necrosis factor receptor-associated factor-1 (TRAF1) with rheumatoid arthritis (RA). CD40 signalling is known to be associated with TRAF1 expression, directly or indirectly; however, the detailed mechanisms of these interactions are not clear in RA, and in particular in fibroblast-like synoviocytes. This study aims to investigate this pathway and the role of nuclear factor-κB (NF-κB) in a mouse model of RA.We utilized the collagen-induced arthritis (CIA) model in DBA/1 mice. CD40, TRAF1, and NF-κB p65 were quantitated by enzyme-linked immunosorbent assay and immunohistochemistry in serum and tissue, respectively. Real-time polymerase chain reaction was applied to measure NF-κB-related gene expression, including cytokines [tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6)] and adhesion molecules [intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1)].The severity of arthritis by clinical and histological assessments peaked on day 35 and decreased thereafter. Serum levels of CD40, TRAF1, and NF-κB p65 paralleled this time-course. The tissue expression of the CD40, TRAF1, total NF-κB p65, and phospho-NF-κB p65 proteins, as well as NF-κB-related gene expression, including cytokines (TNFα, IL-6) and adhesion molecules (ICAM-1, VCAM-1), were markedly upregulated within 25-50 days after CIA.Our data identify a cellular/molecular mechanism of the CD40/TRAF1 signalling pathway involved in CIA: increased expression of CD40 and its adaptor TRAF1 proteins and activation of the NF-κB-dependent proinflammatory pathway. These correlations implicate possible mechanistic pathways in this model that may also operate in human RA and thus provide rationales for new therapeutic modalities.

Helicobacter pylori infection causes the hyper-proliferation of gastric epithelial cells that leads to the development of gastric cancer. Overexpression of tumor necrosis factor receptor associated factor (TRAF) is shown in gastric cancer cells. The dietary antioxidant &beta, carotene has been shown to counter hyper-proliferation in H. pylori-infected gastric epithelial cells. The present study was carried out to examine the &beta, carotene mechanism of action. We first showed that H. pylori infection decreases cellular IB&alpha, levels while increasing cell viability, NADPH oxidase activity, reactive oxygen species production, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) activation, and TRAF1 and TRAF2 gene expression, as well as protein&ndash, protein interaction in gastric epithelial AGS cells. We then demonstrated that pretreatment of cells with &beta, carotene significantly attenuates these effects. Our findings support the proposal that &beta, carotene has anti-cancer activity by reducing NADPH oxidase-mediated production of ROS, NF-B activation and NF-B-regulated TRAF1 and TRAF2 gene expression, and hyper-proliferation in AGS cells. We suggest that the consumption of &beta, carotene-enriched foods could decrease the incidence of H. pylori-associated gastric disorders.

Tumor necrosis factor receptor (TNFR)–associated factor 1 (TRAF1) is a unique TRAF protein that can interact directly or indirectly with multiple TNFR family members, regulatory proteins, kinases, and adaptors that contribute to its diverse functions in specific tissues. However, the role of TRAF1 in non–small cell lung cancer (NSCLC) remains unknown. In this study, we report that TRAF1 is overexpressed in human lung cancer cells and tissues. TRAF1 expression level inversely correlated with patient survival probability. Loss of TRAF1 decelerated tumor invasion in a urethane-induced lung carcinogenesis mouse model. Furthermore, TRAF1 expression affected TRAF2-mediated BRAF Lys48–linked ubiquitination, which was followed by the inhibition of growth and differentiation, and the induction of death in lung cancer cells. Overall, our work suggests that TRAF1 plays a novel role in the regulation of the BRAF/MEK/ERK signaling pathway in NSCLC and offers a candidate molecular target for lung cancer prevention and therapy. Significance: These findings identify TRAF1 as a new therapeutic target for NSCLC. Cancer Res; 78(14); 3982–94. ©2018 AACR.

MicroRNAs are important regulators during human growth and development. Emerging evidence indicates that microRNAs play important roles in colorectal cancer. The aim of this study is to reveal the biological function and direct target gene of miR-483 in colorectal cancer. The biological function of miR-483 on the proliferation and migration of colon cancer cells was then examined by Edu assay and transwell assay, respectively. Our findings revealed that miR-483 mimic could significantly inhibit cell proliferation and migration. The target gene of miR-483 was predicted by target scan software and identified by a dual fluorescence reporter system which showed that TRAF1 was a direct target gene of miR-483 in SW480 cell line. These data suggest that miR-483 is a colorectal cancer suppressor which could inhibit cell proliferation and migration, possibly via targeting TRAF1. The miR-483 could be a potential treatment target for colorectal cancer. Keywords: miR-483, Colon cancer cells, Proliferation, Migration, TRAF1

BackgroundmicroRNA-23b (miR-23b) is a multiple functional miRNA. We hypothesize that miR-23b plays a role in the pathogenesis of sepsis. Our study investigated the effect of miR-23b on sepsis-induced immunosuppression.MethodsMice were treated with miR-23b inhibitors by tail vein injection 2 days after cecal ligation puncture (CLP)–induced sepsis. Apoptosis in spleens and apoptotic signals were investigated, and survival was monitored. T-cell immunoreactivities were examined during late sepsis. Nuclear factor κB (NF-κB)–inducing kinase (NIK), tumor necrosis factor (TNF)–receptor associated factor 1 (TRAF1), and X-linked inhibitor of apoptosis protein (XIAP), the putative targets of miR-23b, were identified by a dual-luciferase reporter assay.ResultsmiR-23b expression is upregulated and sustained during sepsis. The activation of the TLR4/TLR9/p38 MAPK/STAT3 signal pathway contributes to the production of miR-23b in CLP-induced sepsis. miR-23b inhibitor decreased the number of spleen cells positive by terminal deoxynucleotidyl transferase dUTP nick-end labeling and improved survival. miR-23b inhibitor restored the immunoreactivity by alleviating the development of T-cell exhaustion and producing smaller amounts of immunosuppressive interleukin 10 and interleukin 4 during late sepsis. We demonstrated that miR-23b mediated immunosuppression during late sepsis by inhibiting the noncanonical NF-κB signal and promoting the proapoptotic signal pathway by targeting NIK, TRAF1, and XIAP.ConclusionsInhibition of miR-23b reduces late-sepsis-induced immunosuppression and improves survival. miR-23b might be a target for immunosuppression.

Host genetics are important to consider in the role of resistance or susceptibility for developing active pulmonary tuberculosis (TB). Several association studies have reported the role of variants in STAT4 and TRAF1/C5 as risk factors to autoimmune diseases. Nevertheless, more data is needed to elucidate the role of these gene variants in infectious disease. Our data reports for the first time, variant rs10818488 in the TRAF1/C5 gene (found 47% of the population worldwide), is associated with susceptibility (OR = 1.51) to development TB. Multivariate analysis evidenced association between rs10818488 TRAF1/C5 and risk to multibacillary TB (OR = 4.18), confers increased bacteria load in the lung, indicates a decreased ability to control pathogen levels in the lung, and spread of the pathogen to new hosts. We showed that the “loss-of-function” variant in TRAF1/C5 led to susceptibility for TB by decreased production of TNF-α. Our results suggest the role of variant TRAF1/C5 in susceptibility to TB as well as in clinical presentation of multibacillary TB.

Background and objectives: Receptor-interacting serine/threonine-protein kinase-2 (RIPK2) is an important mediator in different pathways in the immune and inflammatory response system. RIPK2 was also shown to play different roles in different cancer types, however, in colorectal cancer (CRC), its role is not well established. This study aims at identifying the role of RIPK2 in CRC progression and survival. Materials and methods: Data of patients and mRNA protein expression level of genes associated with CRC (RIPK2, tumor necrosis factor (TNF), TRAF1, TRAF7, KLF6, interlukin-6 (Il6), interlukin-8 (Il8), vascular-endothelial growth factor A (VEGFA), MKI67, TP53, nuclear factor-kappa B (NFKB), NFKB2, BCL2, XIAP, and RELA) were downloaded from the PrognoScan online public database. Patients were divided between low and high RIPK2 expression and different CRC characteristics were studied between the two groups. Survival curves were evaluated using a Kaplan–Meier estimator. The Pearson correlation was used to study the correlation between RIPK2 and the other factors. Statistical analysis was carried out using SPSS version 25.0. The Human Protein Atlas was also used for the relationship between RIPK2 expression in CRC tissues and survival. Differences were considered statistically significant at p &lt, 0.05. Results: A total of 520 patients were downloaded from the PrognoScan database, and RIPK2 was found to correlate with MKI67, TRAF1, KLF6, TNF, Il6, Il8, VEGFA, NFKB2, BCL2, and RELA. High expression of RIPK2 was associated with high expression of VEGFA (p &lt, 0.01) and increased mortality (p &lt, 0.01). Conclusions: In this study, RIPK2 is shown to be a potential prognostic factor in CRC, however, more studies are needed to assess and verify its potential role as a prognostic marker and in targeted therapy.

Ulcerative colitis (UC), an inflammatory disorder of the colon arises from dysregulated immune response towards gut microbes. Transcription factor NFκB is a major regulatory component influencing mucosal inflammation. We evaluated expression of Tumor Necrosis Factor Alpha Induced Protein3 (TNFAIP3), the inhibitor of NFκB activation and its associated partners ITCH, RNF11 and Tax1BP1 in inflamed mucosa of UC patients. We found highly significant up-regulated mRNA expression of TNFAIP3 that negatively correlated with disease activity in UC. mRNA levels of ITCH, RNF11 and Tax1BP1 were significantly down-regulated. Significant positive correlation with disease activity was noted for Tax1BP1. All four genes showed significant down-regulation at protein level. mRNA levels of inducers of TNFAIP3 expression, NFκB p65 subunit and MAST3 was determined. There was significant increase in p65 mRNA expression and down-regulated MAST3 expression. This suggested that increase in NFκB expression regulates TNFAIP3 levels. Deficiency of TNFAIP3 expression resulted in significant up-regulation of NFκB p65 sub-unit as well as its downstream genes such as iNOS, an inflammatory marker, inhibitors of apoptosis like cIAP2 and XIAP and mediators of anti-apoptotic signals TRAF1 and TRAF2. Taken together, decreased expression of TNFAIP3 and its partners contribute to inflammation and up-regulation of apoptosis inhibitors that may create microenvironment for colorectal cancer.

// Bing Xu 1 , Jian Li 1 , Xiaoxin Liu 2 , Chang Li 3 and Xiaotian Chang 1 1 Medical Research Center of Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan, Shandong, P. R. China 2 Blood Transfusion Department of Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan, Shandong, P. R. China 3 Pathology Department of Tengzhou Central People’s Hospital, Tengzhou, P. R. China Correspondence to: Xiaotian Chang, email: changxt@126.com Keywords: TXNDC5, cervical tumor, SERPINF1, TRAF1, pathway Received: November 24, 2016 Accepted: June 10, 2017 Published: June 29, 2017 ABSTRACT TXNDC5 (thioredoxin domain-containing protein 5) catalyzes disulfide bond formation, isomerization and reduction. Studies have reported that TXNDC5 expression is increased in some tumor tissues and that its increased expression can predict a poor prognosis. However, the tumorigenic mechanism has not been well characterized. In this study, we detected a significant association between the rs408014 and rs7771314 SNPs at the TXNDC5 locus and cervical carcinoma using the Taqman genotyping method. We also detected a significantly increased expression of TXNDC5 in cervical tumor tissues using immunohistochemistry and Western blot analysis. Additionally, inhibition of TXNDC5 expression using siRNA prevented tube-like structure formation, an experimental indicator of vasculogenic mimicry and metastasis, in HeLa cervical tumor cells. Inhibiting TXNDC5 expression simultaneously led to the increased expression of SERPINF1 (serpin peptidase inhibitor, clade F) and TRAF1 (TNF receptor-associated factor 1), which have been reported to inhibit angiogenesis and metastasis as well as induce apoptosis. This finding was confirmed in Caski and C-33A cervical tumor cell lines. The ability to form tube-like structures was rescued in HeLa cells simultaneously treated with anti-TXNDC5, SERPINF1 and TRAF1 siRNAs. Furthermore, the inhibition of TXNDC5 expression significantly attenuated endothelial tube formation, a marker of angiogenesis, in human umbilical vein endothelial cells. The present study suggests that TXNDC5 is a susceptibility gene in cervical cancer, and high expression of this gene contributes to abnormal angiogenesis, vasculogenic mimicry and metastasis by down-regulating SERPINF1 and TRAF1 expression.

TRAF1 is a member of the TRAF protein family, which regulates the canonical and noncanonical NF-κB signaling cascades. Although aberrant TRAF1 expression in tumors has been reported, the role of TRAF1 remains elusive. Here, we report that TRAF1 is required for solar UV-induced skin carcinogenesis. Immunohistochemical analysis showed that TRAF1 expression is up-regulated in human actinic keratosis and squamous cell carcinoma. In vivo studies indicated that TRAF1 expression levels in mouse skin are induced by short-term solar UV irradiation, and a long-term skin carcinogenesis study showed that deletion of TRAF1 in mice results in a significant inhibition of skin tumor formation. Moreover, we show that TRAF1 is required for solar UV-induced extracellular signal-regulated kinase–5 (ERK5) phosphorylation and the expression of AP-1 family members (c-Fos/c-Jun). Mechanistic studies showed that TRAF1 expression enhances the ubiquitination of ERK5 on lysine 184, which is necessary for its kinase activity and AP-1 activation. Overall, our results suggest that TRAF1 mediates ERK5 activity by regulating the upstream effectors of ERK5 and also by modulating its ubiquitination status. Targeting TRAF1 function might lead to strategies for preventing and treating skin cancer.

Cytotoxin-associated gene A (CagA) is one of the most important virulence factors of Helicobacter pylori, and serves a role in H. pylori‑mediated tumorigenesis in gastric cancer. However, the underlying molecular mechanism remains to be elucidated. The present study aimed to investigate the effects of CagA on the proliferation and apoptosis of GES‑1 cells, and the underlying mechanism. A CagA eukaryotic expression plasmid was constructed and transfected into GES‑1 cells. The mRNA and protein levels of CagA, tumor necrosis factor receptor‑associated factor 1 (TRAF1) and tumor necrosis factor receptor superfamily member 9 (4‑1BB) were determined using the reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. Western blot and ELISA analysis was used to detect the release of interleukin (IL)‑8. An MTT assay and flow cytometric analysis was used to assess cell viability and apoptosis, respectively. Ectopic expression of CagA markedly increased TRAF1 and 4‑1BB mRNA and protein levels in GES‑1 cells. CagA increased the expression and release of IL‑8 in GES‑1 cells. The expression of CagA significantly promoted the proliferation (P

Background After acute myocardial infarction, the recovery of ischemic myocardial blood flow may cause myocardial reperfusion injury, which reduces the efficacy of myocardial reperfusion. Ways to reduce and prevent myocardial ischemia/reperfusion (I/R) injury are of great clinical significance in the treatment of patients with acute myocardial infarction. TRAF1 (tumor necrosis factor receptor–associated factor 1) is an important adapter protein that is implicated in molecular events regulating immunity, inflammation, and cell death. Little is known about the role and impact of TRAF 1 in myocardial I/R injury. Methods and Results TRAF 1 expression is markedly induced in wild‐type mice and cardiomyocytes after I/R or hypoxia/reoxygenation stimulation. I/R models were established in TRAF 1 knockout mice and wild type mice (n=10 per group). We demonstrated that TRAF 1 deficiency protects against myocardial I/R–induced loss of heat function, inflammation, and cardiomyocyte death. In addition, overexpression of TRAF 1 in primary cardiomyocytes promotes hypoxia/reoxygenation‐induced inflammation and apoptosis in vitro. Mechanistically, TRAF 1 promotes myocardial I/R injury through regulating ASK1 (apoptosis signal‐regulating kinase 1)–mediated JNK/p38 (c‐Jun N‐terminal kinase/p38) MAPK (mitogen‐activated protein kinase) cascades. Conclusions Our results indicated that TRAF 1 aggravates the development of myocardial I/R injury by enhancing the activation of ASK 1‐mediated JNK /p38 cascades. Targeting the TRAF 1– ASK 1– JNK /p38 pathway provide feasible therapies for cardiac I/R injury.

Alongside with the emergent clinical trials for Machado-Joseph disease/Spinocerebellar ataxia type 3 (MJD/SCA3) comes the need to identify molecular biomarkers of disease that can be tracked throughout the trial. MJD is an autosomal dominant neurodegenerative disorder caused by expansion of a CAG repeat in the coding region of the ATXN3 gene. Previous findings indicate the potential of transcriptional alterations in blood of MJD patients as biomarkers of disease. Accurate quantification of gene expression levels by quantitative real-time PCR (qPCR) depends on data normalization, usually performed using reference genes. Because the expression level of routinely used housekeeping genes may vary in multiple biological and experimental conditions, reference gene controls should be validated in each specific experimental design. Here, we aimed to evaluate the expression behavior of five housekeeping genes previously reported as stably expressed in peripheral blood of patients from several disorders-peptidylprolyl isomerase B (PPIB), TNF receptor associated protein 1 (TRAP1), beta-2-microglobulin (B2M), 2,4-dienoyl-CoA reductase 1 (DECR1), and folylpolyglutamate synthase (FPGS). Expression levels of these five genes were assessed by qPCR in blood from MJD subjects (preataxic and patients) and matched controls. While all housekeeping genes, here studied, were stably expressed in our sets of samples, TRAP1 showed to be the most stable gene by NormFinder and BestKeeper. We, therefore, conclude that any of these genes could be used as reference gene in future qPCR studies using blood samples from MJD subjects.

EBV-associated gastric carcinoma (EBVaGC) is a specific subgroup of gastric carcinoma, and the multifunctional transcriptional factor NF-κB may contribute to its tumorigenesis. In this study, we comprehensively characterized NF-κB signaling in EBVaGC using qRT-PCR, western blot, immunofluorescence assays, ELISA, and immunohistochemistry staining. NF-κB-signaling inhibitors may inhibit the growth of EBVaGC cells and induce significant apoptosis. IκBα is a key regulatory molecule, and repression of IκBα can contribute to aberrant NF-κB activation. Overexpression of LMP1 and LMP2A in the EBV-negative GC cell line SGC7901 could inhibit the expression of IκBα and induce NF-κB activation. These findings indicate that the canonical NF-κB signal is constitutively activated and plays an important role in EBVaGC tumorigenesis.

Natural Killer (NK) cells control metastatic dissemination of murine tumors and are an important prognostic factor in several human malignancies. However, tumor cells hijack many of the NK cell functional features compromising their tumoricidal activity. Here, we show a deleterious role of the TNFα/TNFR2/BIRC3/TRAF1 signaling cascade in NK cells from the tumor microenvironment (TME). TNFα induces BIRC3/cIAP2 transcripts and reduces NKp46/NCR1 transcription and surface expression on NK cells, promoting metastases dissemination in mice and poor prognosis in GIST patients. NKp30 engagement, by promoting the release of TNFα, also contributes to BIRC3 upregulation, and more so in patients expressing predominantly NKp30C isoforms. These findings reveal that in the absence of IL-12 or a Th1-geared TME, TNFα can be considered as a negative regulatory cytokine for innate effectors.

Background Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a member of the TRAF family and is dysregulated in diseases, such as atheroma, lymphoma, and solid tumors, but the role of TRAF1 in gastric cancer remains unknown. This study was aimed to investigate the role of TRAF1 in Helicobacter pylori (H. pylori)-related cell apoptosis and gastric carcinogenesis. Materials and methods The mRNA and protein expression levels of TRAF1 were measured in a panel of gastric cancer cell lines and in H. pylori -infected mice by quantitative real-time PCR (qPCR) and Western blotting. The transcription factor that mainly affects transcription of TRAF1 during H. pylori infection was identified. The roles of H. pylori virulence factors that regulate TRAF1 expression were analyzed using isogenic cagA-, vacA-, and cagE-null mutants. The effects of TRAF1 on gastric cell viability and apoptosis during H. pylori infection were detected using the standard MTS (cell viability) assay and flow cytometry, respectively. Results H. pylori infection induced TRAF1 overexpression in both gastric epithelial cells and mice. The expression of TRAF1 in response to H. pylori infection was majorly regulated by the activation of NF-κB and was strongly related to H. pylori virulence factor CagA. The upregulation of TRAF1 inhibited cell apoptosis and increased the viability of infected cells. Conclusions H. pylori infection induces the overexpression of TRAF1 in gastric epithelial cells. The upregulation of TRAF1 plays an antiapoptotic role in H. pylori -infected gastric cells and may contribute to the gastric carcinogenesis.

Aim of the study Recent studies demonstrated the association of tumor necrosis factor α-induced protein 3 (TNFAIP3) (rs2230926) and tumor necrosis factor receptor associated factor 1 (TRAF1) (rs10818488) with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in different populations. We aimed at determining whether they confer susceptibility to SLE and RA in Egyptian population and if there is any relation to disease activity and auto-antibodies profile. Material and methods A case-control study involving 105 individuals with RA, 90 with SLE and 75 healthy controls was performed using TaqMan genotyping assay for two SNPs that showed the best evidence of association in the previous Caucasian studies. Results We detected significant differences in G allele frequency of TNFAIP3 (rs2230926) with SLE (p = 0.017*) and RA (OR = 2.333; 95% CI: 1.103-4.935, p = 0.023*) and association with RA disease activity (< 0.001). The A allele of TRAF1 was significantly increased in RA compared to controls(p = 0.049) and with RA activity (p = 0.001), while TRAF1 polymorphism did not exhibit any significant difference in the frequencies of genotypes or alleles in SLE and control (p = 0.280). Conclusions TNFAIP3 is a susceptibility gene to SLE and RA in the Egyptian population and is correlated to disease activity and the presence of autoantibodies while TRAF1 polymorphisms increase the risk of RA but not to SLE in Egyptian populations.

Objective To study the expression of tumor necrosis factor receptor-associated protein 1 (TRAP1) in gastric cancer tissue and its correlation with malignant biology. Methods Gastric cancer tissue and adjacent normal tissue were collected, and mRNA content and protein content of TRAP1 were detected; gastric cancer cell lines SGC7901, BGC823, AGS and MGC803 were cultured, and mRNA contents and protein contents of TRAP1, CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF were detected. Results mRNA and protein expression levels of TRAP1 in gastric cancer tissue were significantly higher than those in adjacent normal tissue, and mRNA and protein expression levels of TRAP1 in gastric cancer tissue with muscularis and serosa infiltration, lymph node metastasis, distant organ metastasis and TNM Ⅲ/Ⅳ stage were significantly higher than those in gastric cancer tissue with mucosa and submucosa infiltration, non-lymph node metastasis, non-distant organ metastasis and TNM Ⅰ/Ⅱ stage. mRNA and protein expression levels of TRAP1, CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF in MGC803 were the highest, and mRNA and protein expression levels of TRAP1, CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF in SGC7901 were the lowest. mRNA and protein expression levels of TRAP1 in gastric cancer cell lines were positively correlated with mRNA and protein expression of CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF. Conclusions The expression of TRAP1 significantly increases in gastric cancer tissue; TRAP1 may regulate the malignant biology of cells by increasing the expression of CyclinB1, CyclinD1, CyclinE, MMP-2 and VEGF, thereby resulting in the occurrence and development of gastric cancer.

CD137 (4-1BB, Tnsfr9) is a member of the TNF-receptor (TNFR) superfamily without known intrinsic enzymatic activity in its cytoplasmic domain. Hence, akin to other members of the TNFR family, it relies on the TNFR-Associated-Factor (TRAF) family of adaptor proteins to build the CD137 signalosome for transducing signals into the cell. Thus, upon CD137 activation by binding of CD137L trimers or by crosslinking with agonist monoclonal antibodies, TRAF1, TRAF2, and TRAF3 are readily recruited to the cytoplasmic domain of CD137, likely as homo- and/or heterotrimers with different configurations, initiating the construction of the CD137 signalosome. The formation of TRAF2-RING dimers between TRAF2 molecules from contiguous trimers would help to establish a multimeric structure of TRAF-trimers that is probably essential for CD137 signaling. In addition, available studies have identified a large number of proteins that are recruited to CD137:TRAF complexes including ubiquitin ligases and proteases, kinases, and modulatory proteins. Working in a coordinated fashion, these CD137-signalosomes will ultimately promote CD137-mediated T cell proliferation and survival and will endow T cells with stronger effector functions. Current evidence allows to envision the molecular events that might take place in the early stages of CD137-signalosome formation, underscoring the key roles of TRAFs and of K63 and K48-ubiquitination of target proteins in the signaling process. Understanding the composition and fine regulation of CD137-signalosomes assembly and disassembly will be key to improve the therapeutic activities of chimeric antigen receptors (CARs) encompassing the CD137 cytoplasmic domain and a new generation of CD137 agonists for the treatment of cancer., Funding was from Instituto de Salud Carlos III (ISCIII) (PI16/00895 to JZ), Ministerio de Economía y Competitividad (MINECO) RTC-16-5118-1 to JZ and IM, SAF 2014-52361-R and SAF 2017-83267-C2-1R to IM; and Cancer Research Institute (CRI) CLIP Grant 2017, Asociación Española Contra el Cancer, Fundación BBVA and I-ON Network-BMS to IM. The cost of this publication was paid in part by funds from the European Fund for Economic and Regional Development (FEDER).

Exploring effective therapies for delaying calcific heart valve disease (CHVD) is the focus of current cardiovascular clinical and scientific research. In this study, human aortic valve interstitial cells (hVICs) were isolated from patients with CHVD. After expansion, cultured hVICs were induced with the tumor necrosis factor-alpha (TNF-α) with or without anthraquinone emodin (EMD) treatments. Cytotoxicity and flow cytometric analysis were used to assess cell growth, while Alizarin Red S staining was used to detect hVICs calcification. Furthermore, RNA-sequencing analysis was utilized to investigate changes in mRNA profiles of cells cultured in TNF-α conditioned medium with or without EMD. Western blotting and qRT-PCR analyses were used for the verification of key selected genes. Our results indicated that EMD had limited influence on hVIC morphology, whereas in a dose-dependent fashion, EMD interfered with hVIC growth under TNF-α conditioned cell culture. Additionally, hVICs treated with TNF-α plus EMD, presented a gradual decrease of positive Alizarin Red S staining, when compared with cells treated with TNF-α only. Notably, cells treated with TNF-α plus EMD showed 1874 differential expression genes (DEGs), among them, 1131 were upregulated and 743 were downregulated. These DEGs displayed an enrichment of biological functions and signaling pathways, among them, BMP2, RELA, TNF, and TRAF1, were found to be significantly suppressed by EMD and selected given their role in mediating hVIC calcification. In conclusion, our study shows that EMD inhibits TNF-α-induced calcification and phenotypical transformation of hVICs via the NF-κB signaling pathway, thereby preventing calcification events stimulated during acute inflammatory responses.

This study investigated the role of miR-214 in the hepatocyte apoptosis induced by hypoxia/reoxygenation (H/R) injury. In vivo hepatic ischemia/reperfusion (HIR) injury, mice model and in vitro HR model were established. miR-214, TRAF1, ASK1, and JNK expression levels were detected by qRT-PCR and western blot. The apoptosis of mouse hepatocyte AML12 was detected by flow cytometry analysis. The interaction between miR-214 and TRAF1 was confirmed by dual-luciferase reporter gene assay. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were elevated in HIR injury mice compared with sham mice. miR-214 expression was down-regulated in liver tissues of HIR and H/R-induced hepatocytes, whereas TRAF1, ASK1, and JNK expressions were up-regulated in HIR and H/R groups. H/R stimulation promoted the apoptosis of hepatocytes, and miR-214 overexpression inhibited the apoptosis of hepatocytes. Besides, TRAF1 was a target of miR-214 and negatively regulated by miR-214. miR-214/TRAF1 pathway involved in the modulation of H/R-induced apoptosis of hepatocytes. In vivo study proved miR-214 reduced hepatic injury of HIR mice. miR-214 overexpression reduces hepatocyte apoptosis after HIR injury through negatively regulating TRAF1/ASK1/JNK pathway.

Chimeric antigen receptors (CARs) have an antigen-binding domain fused to transmembrane, costimulatory, and CD3ζ domains. Two CARs with regulatory approval include a CD28 or 4-1BB costimulatory domain. While both CARs achieve similar clinical outcomes, biologic differences have become apparent but not completely understood. Therefore, in this study we aimed to identify mechanistic differences between 4-1BB and CD28 costimulation that contribute to the biologic differences between the 2 CARs and could be exploited to enhance CAR T cell function. Using CD19-targeted CAR T cells with 4-1BB we determined that enhancement of T cell function is driven by NF-κB. Comparison to CAR T cells with CD28 also revealed that 4-1BB is associated with more antiapoptotic proteins and dependence on persistence for B cell killing. While TNF receptor–associated factor 2 (TRAF2) has been presupposed to be required for 4-1BB costimulation in CAR T cells, we determined that TRAF1 and TRAF3 are also critical. We observed that TRAFs impacted CAR T viability and proliferation, as well as cytotoxicity and/or cytokines, in part by regulating NF-κB. Our study demonstrates how 4-1BB costimulation in CAR T cells impacts antitumor eradication and clinical outcomes and has implications for enhanced CAR design.

Background: miR-218–5p is an extensively studied microRNA (miRNA) in prostate cancer (PCa). However, the clinical significance and biological role of miR-218–5p in bone metastasis of PCa remain unclear. Materials and Methods: miR-218–5p expression was evaluated in 38 bone metastatic and 115 non-bone metastatic PCa tissues and serum samples. Clinical correlation of miR-218–5p expression with clinicopathological characteristics was analyzed. The biological roles of miR-218–5p in bone metastasis of PCa were investigated in vitro by invasion and migration assays. Bioinformatics analysis, real-time polymerase chain reaction, western blot, and luciferase reporter assay were applied to discern and examine the relationship between miR-218–5p and its potential targets. Results: miR-218–5p expression was reduced in bone metastatic PCa tissue and serum samples, which positively correlated with poor clinicopathological characteristics and bone metastasis-free survival in PCa patients. Upregulating miR-218–5p repressed PCa cell invasion and migration. Furthermore, miR-218–5p inhibited NF-κB signaling via simultaneously targeting TRAF1, TRAF2, and TRAF5, which suppressed the invasion and migration abilities of PCa cells. ROC curve analysis of miR-218–5p in the serum of PCa patients exhibited an area under the curve of 0.86 (95% confidence interval 0.80–0.92, p Conclusion: Our findings indicate that miR-218–5p might represent a novel serum biomarker for bone metastasis of PCa.

Oral leukoplakia (OL) is one of the most common oral precancerous lesions with the possibility of malignant transformation, ranging from 17 to 24% of patients with a median follow-up of >7 years. Previous research has revealed that compared with normal oral epithelial tissues, the expression of secretory leukocyte peptidase inhibitor (SLPI) protein is significantly reduced in oral squamous cell carcinoma (OSCC). Based on the above-mentioned research, it is known that SLPI is a potential predictive and diagnostic tool for the progression of oral carcinogenesis. Therefore, we investigated the correlation between the abundance of SLPI protein and the different histological grades of OL by immunohistochemistry. The results indicated that the level of SLPI was negatively correlated with the histological grades of the oral premalignant lesions, indicating that it may be a potential predictive tool for the malignant transformation presented in oral precancerous patients. Subsequently, we investigated the biological effects of SLPI using Cell Counting Kit (CCK)-8, Annexin V/PI apoptosis assay and Caspase-Glo® 3/7 assay. The findings revealed that SLPI promoted apoptosis in the Leuk1 and WSU-HN4 cell lines. Mechanistic studies indicated that SLPI, at least in part, regulated cell apoptosis by inhibiting the expression of TNF receptor-associated factor 1 (TRAF1), which has a close relationship with the nuclear factor-κB (NF-κB) pathway.

Background/Aims: Accumulated evidence indicates that lncRNA NEAT1 has important roles in various malignant tumors. In this study, we conducted a comprehensive analysis to explore the exact role of NEAT1 in hepatocellular carcinoma (HCC). Methods: The effects of NEAT1 on cell proliferation, apoptosis, migration, and invasion were measured by in vitro experiments. The expression level and clinical value of NEAT1 in HCC was evaluated based on data from The Cancer Genome Atlas (TCGA), Oncomine, and in-house real-time quantitative (RT-qPCR). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein-protein interaction (PPI) network analyses were conducted to investigate the potential molecular mechanisms of NEAT1. Results: NEAT1 siRNA not only inhibited proliferation, migration, and invasion of HCC cells but also induced HCC cell apoptosis. A total of four records from TCGA, Oncomine, and RT-qPCR analysis were combined to assess the expression level of NEAT1 in HCC. The pooled standard mean deviation (SMD) indicated that NEAT1 was up-regulated in HCC (SMD = 0.54; 95% CI, 0.36–0.73; P < 0.0001). The area under the curve value of the summary receiver operating characteristic curve was 0.71. NEAT1 expression was also related to race (P = 0.025) and distant metastasis (P = 0.002). Additionally, the results of GO, KEGG pathway, and PPI network analyses suggest that NEAT1 may promote the progression of HCC by interacting with several tumor-related genes (SP1, MDM4, CREBBP, TRAF5, CASP8, TRAF1, KAT2A, and HIST4H4). Conclusions: NEAT1 contributes to the deterioration of HCC and provides a potential biomarker for the diagnosis and therapy of HCC.

Hepatitis C virus (HCV)-specific CD8+ T cells suffer a progressive exhaustion during persistent infection (PI) with HCV. This process could involve the positive immune checkpoint 4-1BB/4-1BBL through the loss of its signal transducer, TRAF1. To address this issue, peripheral HCV-specific CD8+ T cells (pentamer-positive [pentamer+]/CD8+ T cells) from patients with PI and resolved infection (RI) after treatment were studied. The duration of HCV infection and the liver fibrosis progression rate inversely correlated with the likelihood of detection of peripheral pentamer+/CD8+ cells. In PI, pentamer+/CD8+ cells had impaired antigen-specific reactivity that worsened when these cells were not detectable ex vivo Short/midduration PI was characterized by detectable peripheral PD-1+ CD127low TRAF1low cells. After triggering of T cell receptors (TCR), the TRAF1 level positively correlated with the levels of CD127, Mcl-1, and CD107a expression and proliferation intensity but negatively with PD-1 expression, linking TRAF1low to exhaustion. In vitro treatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-β1 (TGF-β1) did the opposite, suggesting that the IL-7/TGF-β1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-β1 concentration was higher in patients with PI than in patients with RI, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing the level of TRAF1 expression, IL-7 plus 4-1BBL treatment in vitro enhanced T cell reactivity in patients with short/midduration infection. However, in patients with long-lasting PI, anti-PD-L1, in addition to the combination of IL-7 and 4-1BBL, was necessary to reestablish T cell proliferation in individuals with slowly progressing liver fibrosis (slow fibrosers) but had no effect in rapid fibrosers. In conclusion, a peripheral hyporeactive TRAF1low HCV-specific CD8+ T cell response, restorable by IL-7 plus 4-1BBL treatment, characterizes short/midduration PI. In long-lasting disease, HCV-specific CD8+ T cells are rarely detectable ex vivo, but treatment with IL-7, 4-1BBL, and anti-PD-L1 recovers their reactivity in vitro in slow fibrosers.IMPORTANCE Hepatitis C virus (HCV) infects 71 million people worldwide. Two-thirds develop a chronic disease that can lead to cirrhosis and hepatocellular carcinoma. Direct-acting antivirals clear the infection, but there are still patients who relapse. In these cases, additional immunotherapy could play a vital role. A successful anti-HCV immune response depends on virus-specific CD8+ T cells. During chronic infection, these cells are functionally impaired, which could be due to the failure of costimulation. This study describes exhausted specific T cells, characterized by low levels of expression of the signal transducer TRAF1 of the positive costimulatory pathway 4-1BB/4-1BBL. IL-7 upregulated TRAF1 expression and improved T cell reactivity in patients with short/midduration disease, while in patients with long-lasting infection, it was also necessary to block the negative PD-1/PD-L1 checkpoint. When the results are taken together, this work supports novel ways of restoring the specific CD8+ T cell response, shedding light on the importance of TRAF1 signaling. This could be a promising target for future immunotherapy.

BACKGROUND TNFR-associated factor 1 (TRAF1) and TRAF2 have been demonstrated to inhibit apoptosis and promote cell survival in glioblastoma (GBM) cells with experiments in vitro. However, their clinical and prognostic significance have not been elucidated. MATERIAL AND METHODS In our study, we for the first time investigated the expression of TRAF1 and TRAF2 in 105 GBM tissues. Furthermore, we evaluated their clinical significance, including their association with clinicopathologic factors and prognostic value. The association with clinicopathologic factors was assessed by chi-square test. The relation of TRAF1/2 expression to survival rate was assessed by Kaplan-Meier method and Cox-regression model. RESULTS We demonstrated that TRAF1 expression had no significant prognostic value for GBM. On the contrary, high expression of TRAF2 can predict poorer prognosis of GBM and was identified as an independent biomarker in GBM prognosis. CONCLUSIONS High expression of TRAF2 was identified as an independent biomarker in GBM prognosis, indicating TRAF2 as a novel drug target in GBM treatment.

Background and aim Non-small cell lung cancer (NSCLC), which comprises 80%-85% of all lung cancer cases, is one of the most common human malignancies. Despite great improvements in diagnostic technology and the introduction of new therapeutic agents in recent years, the 5-year survival rate of NSCLC is still low. Tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1) plays an important role in the TNF-related apoptosis-inducing ligand (TRAIL) associated signal pathway. Methods In this study, we aim to illuminate the function of TRAF1 in NSCLC. Toward that end, TRAF1 expression was detected using immunohistochemistry (IHC) in specimens from 200 NSCLC patients. The function of TRAF1 in the A549 and H1299 cell lines was evaluated by colony formation and MTT assays. Results Our data showed that TRAF1 was significantly upregulated in NSCLC tissues. TRAF1 expression was positively associated with NSCLC lymphatic metastasis and clinical stage and was negatively associated with overall patient survival. TRAF1 promoted NSCLC cell proliferation CONCLUSION: TRAF1 expression was positively associated with NSCLC lymphatic metastasis and histological grade and was negatively associated with overall patient survival. TRAF1 may be an important therapeutic target for NSCLC.

Background Polymorphisms in the TRAF1-C5 locus have been associated with susceptibility to rheumatoid arthritis (RA). A single nucleotide polymorphism (SNP) rs3761847, located in the first intron of the TRAF1 gene, has been identified as an RA risk locus in a genome-wide association study (GWAS); the relationship of rs3761847 with joint damage are unclear. One of the key mechanisms of joint destruction is the degradation of and invasion of cartilage by RA synovial fibroblasts (RASF). Objectives To determine whether the expression of TRAF1-C5 genes and the invasion of RASF are associated with the genotype at rs3761847. Methods The genotype at rs3761847 was determined using TaqMan Allelic Discrimination Assay. Invasion assays were performed using Matrigel-coated invasion chambers. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation. Neutrophils were isolated from the red blood cell layer by erythrocyte lysis; CD14+ cells were separated using magnetic cell sorting. Macrophages were differentiated from plastic-adhered monocytes in M-CSF (50ng/ml) for 8 days. Cells were stimulated with lipopolysaccharide (LPS, 100ng/ml) plus interferon γ (IFNγ, 20ng/ml), interleukin 4 (IL-4, 20ng/ml) or tumour necrosis factor α (TNF-α, 10ng/ml). Gene expression was measured by SYBR Green real-time PCR using HPRT1 as a housekeeping gene. Results rs3761847 was in Harvey-Weinberg equilibrium in RASF (n=54, p=0.98) with genotype frequencies of 0.42 AA, 0.45 AG and 0.13 GG. When grouped by genotype, the expression of TRAF1 (total), TRAF1 transcript variant 2 and complement C5 mRNA showed no significant difference (p=0.929, p=0.583, p=0.980 respectively). This was also the case for invasion of RASF (n=43, p=0.548; mean cell count 10.4 AA, 8.3 AG and 11.1 GG). There was no correlation of genotype with common RA disease markers (CRP p=0.718, ESR p=0.179, RF titre p=0.466, ACPA titre p=0.712). In contrast, analyzing TRAF1 expression in different types of blood cells, significantly higher levels of TRAF1 mRNA were detected in monocytes carrying the AA genotype compared with the GG genotype at rs3761847, both in unstimulated (2.26-fold) as well as LPS+IFNγ-activated cells (2.04-fold; p Conclusions Our findings suggest that there is no relationship between invasive capacity of RASF or expression of TRAF1-C5 genes and genotype at rs3761847. In contrast, we report an association of the rs3761847 genotype and TRAF1 expression in monocytes. These data underline the importance of studying genotype-phenotype associations in the different cell types relevant for RA pathogenesis. Disclosure of Interest None declared

Background Rheumatoid Arthritis (RA) is the most common systemic autoimmune disease, with a respective expanded genetic research1. Immunogenetic studies have documented the positive correlation of various gene loci with incidence and/or disease profile. However, the description of gene loci negatively related to the incidence of RA is rarely documented. Apart from an early study involving HLA class II, there has been no reference to any genetic locus associated with a protective role against RA incidence. Objectives To identify the sequence of the functional areas of the TRAF1 (TNF receptor associated factor 1 - a protein involved in the intracellular signaling pathway of TNF) gene. Methods 172 patients and 95 controls were genetically assessed for the sequence of the seven exons of the gene TRAF1. Results On the position 9:120905076 of exon 7, the registered polymorphism G/A (rs143265058) was described in the controls group. The same polymorphism was not confirmed in any of the patients. Further functional proteomic study of the polymorphism with computing programs (software), revealed that the presence of this polymorphism leads to a differentiation of the quaternary structure of TRAF1 protein, possibly affecting the cohesion of intracellular TNF signaling pathway2. Conclusions The present reference is one of the extremely rare genetic studies describing a protective gene locus against rheumatoid arthritis, and a pioneer of its kind in the use of Applied Informatics in the depiction of the quaternary structure of the encoded protein. At the same time, it is one of the few immunogenetic studies describing the functional proteomics of the encoded protein, plotting on a molecular level specific interaction modifications affecting the intracellular signaling pathway of TNF. References Kim K, Bang SY, Lee HS, Bae SC. Update on the genetic architecture of rheumatoid arthritis. Nat Rev Rheumatol. 2017 Jan; 13 (1): 13–24. A. Sarantopoulos. TRAF-1 immunogenic study in Rheumatoid Arthritis. PhD Thesis, Aristotle University of Thessaloniki, 2016. (Http://thesis.ekt.gr/thesisBookReader/id/38410#page/1/mode/2up). Disclosure of Interest None declared

In the present study, the aim was to investigate the role of microRNA-1180 (miR-1180) in the growth and apoptosis of prostate cancer, as well as to identify its direct targets. Initially, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to examine the expression of miR-1180 in the prostate cancer tissues and adjacent normal prostate tissues of 30 patients, as well as in DU145 and RWPE-1 cells. Next, DU145 cells were transfected with miR-1180 mimics, and the expression levels of associated proteins were determined by western blot assay. In addition, the role of miR-1180 in the proliferation, apoptosis, invasion and migration of DU145 cells was investigated by MTT, flow cytometry, cell invasion and wound healing assays, respectively. A dual-luciferase reporter assay was also performed to examine whether TNF receptor associated factor 1 (TRAF1) and B-cell lymphoma-2-associated athanogene 2 (BAG2) are direct targets of miR-1180. It was observed that miR-1180 expression was significantly decreased in the prostate cancer tissues compared with the normal prostate tissues, and was also inhibited in DU145 cells compared with RWPE-1 cells. Furthermore, transient overexpression of miR-1180 inhibited the proliferation, migration and invasion, and promoted the apoptosis of DU145 cells, as well as alleviated expression of associated proteins. The dual-luciferase reporter assay confirmed that TRAF1 and BAG2 are direct targets of miR-1180. These results suggested that miR-1180 contributed to prostate cancer by targeting TRAF1/BAG2 and by nuclear factor-κB signaling pathway activation.

Tumor necrosis factor receptor–associated factor 1, an adaptor protein of tumor necrosis factor 2, is involved in classical nuclear factor (NF)-κB activation and lymphocyte recruitment. However, less is known about the expression and association of tumor necrosis factor receptor–associated factor 1 with cancer stem cell markers in oral squamous cell carcinoma. This study aimed to investigate the expression of tumor necrosis factor receptor–associated factor 1 and stem cell characteristic markers (lin28 homolog B, B cell-specific Moloney murine leukemia virus integration site 1, and aldehyde dehydrogenase 1) in oral squamous cell carcinoma and analyze their relations. Paraffin-embedded tissues of 78 oral squamous cell carcinomas, 39 normal oral mucosa, and 12 oral dysplasia tissues were employed in tissue microarrays, and the expression of tumor necrosis factor receptor–associated factor 1, B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B was measured by immunohistostaining and digital pathological analysis. The expression of tumor necrosis factor receptor–associated factor 1 was higher in the oral squamous cell carcinoma group as compared with the expression in the oral mucosa (p 2 = 0.109; p 2 = 0.64; and p 2 = 0.16) in oral squamous cell carcinoma. The patient survival rate was lower in the highly expressed tumor necrosis factor receptor–associated factor 1 group, although the difference was not significant. The clustering analysis showed that tumor necrosis factor receptor–associated factor 1 was most related to aldehyde dehydrogenase 1. These findings suggest that tumor necrosis factor receptor–associated factor 1 has potential direct/indirect regulations with the cancer stem cell markers in oral squamous cell carcinoma, which may help in further analysis of the cancer stem cell characteristics.

Background Recently, epigenetics has become an important field of research, with several diseases related to alteration in methylation patterns. Whether these epimutations are early events due to a local perturbation causing the initial epigenetic event and then leading to pathogenesis, or a later consequence of the pathology itself remains open for discussion. The objective of this study is to assess the methylation patterns of CpG islands in naive and memory T-cells and monocytes from drug-naive, early Rheumatoid arthritis (RA) patients. Materials and methods The methylation patterns of 480,000 CpGs were measured in 6 healthy controls and 10 RA patients using an Illumina methylation genome-wide array (EWAS). T-tests were used to calculate the significance of the methylation differences at each CpG site. A p-value of 1 × 10–4 was used to elucidate highly specific differences. Methylation patterns of individual promoters were then reconstructed using the Human genome database for the position and length of CpG islands. The genes with 3 or more differentially methylated CpGs clustered over a short distance and localised in potentially regulatory regions of the gene were deemed relevant. Results Analysis of the EWAS data showed 4123 CpG sites differently methylated in naive T-cells, 2672 in memory T-cells and 904 in monocytes. Of the relevant genes, TNFR1 appeared particularly interesting. We therefore focused our next analysis on the TNF-α signalling pathway as this has proven pivotal in early disease. Results highlight 9 genes implicated in the 3 TNF-signalling cascades: TNFR1, LTBR, TRAF1, BCL2, UACA, RNF36, MIR21, DAXX and MAP3K14-AS1. These 9 loci exhibited different methylation patterns in naive T-cells. Each had between 3 and 10 CpGs with differential methylation over short DNA regions ( Conclusion Naive T-cells have been implicated in RA pathogenesis through activation/differentiation by cytokine rather than antigen.1 This data confirms the selective role of naive T-cells in early RA primarily linked to differences in up- and down-regulation of elements involved in the TNF-signalling pathway. Reference Blood, 2002;100:4550–6.

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent extrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. However, the mechanisms that contribute to tumor metastasis remain unclear. Here we evaluate the effects of ATPase inhibitory factor 1 (IF1), an inhibitor of the mitochondrial H(+)-adenosine triphosphate (ATP) synthase, on HCC angiogenesis and metastasis. We found that increased expression of IF1 in human HCC predicts poor survival and disease recurrence after surgery. Patients with HCC who have large tumors, with vascular invasion and metastasis, expressed high levels of IF1. Invasive tumors overexpressing IF1 were featured by active epithelial-mesenchymal transition (EMT) and increased angiogenesis, whereas silencing IF1 expression attenuated EMT and invasion of HCC cells. Mechanistically, IF1 promoted Snai1 and vascular endothelial growth factor (VEGF) expression by way of activating nuclear factor kappa B (NF-κB) signaling, which depended on the binding of tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1) to NF-κB-inducing kinase (NIK) and the disruption of NIK association with the TRAF2-cIAP2 complex. Suppression of the NF-κB pathway interfered with IF1-mediated EMT and invasion. Chromatin immunoprecipitation assay showed that NF-κB can bind to the Snai1 promoter and trigger its transcription. IF1 was directly transcribed by NF-κB, thus forming a positive feedback signaling loop. There was a significant correlation between IF1 expression and pp65 levels in a cohort of HCC biopsies, and the combination of these two parameters was a more powerful predictor of poor prognosis. Conclusion: IF1 promotes HCC angiogenesis and metastasis by up-regulation of Snai1 and VEGF transcription, thereby providing new insight into HCC progression and IF1 function. (Hepatology 2014;60:1659–1673)