Bladder cancer (BLCA) is a common genitourinary cancer in patients, and tumour angiogenesis is indispensable for its occurrence and development. However, the indepth mechanism of tumour angiogenesis in BLCA remains elusive. According to recent studies, the tight junction protein family member occludin (OCLN) is expressed at high levels in BLCA tissues and correlates with a poor prognosis. Downregulation of OCLN inhibits tumour angiogenesis in BLCA cells and murine xenografts, whereas OCLN overexpression exerts the opposite effect. Mechanistically, the RT-qPCR analysis and Western blotting results showed that OCLN increased interleukin-8 (IL8) and p-signal transducer and activator of transcription 3 (STAT3) levels to promote BLCA angiogenesis. RNA sequencing analysis and dual-luciferase reporter assays indicated that OCLN regulated IL8 transcriptional activity via the transcription factor STAT4. In summary, our results provide new perspectives on OCLN, as this protein participates in the development of BLCA angiogenesis by activating the IL8/STAT3 pathway via STAT4 and may serve as a novel and unique therapeutic target.

Bladder cancer (BLCA) is the most common malignant tumor of the urinary system and is characterized by high metastatic rates and poor prognosis. The expression of tight junction protein 1 (TJP1) is associated with bladder cancer invasion; however, the mechanism by which TJP1 affects vasculature remodeling remains unknown. In this study, we found that TJP1 expression correlated with tumor angiogenesis and poor overall survival in clinical samples. Furthermore, TJP1 overexpression promoted tumor angiogenesis in BLCA cells and stimulated recruitment of macrophages to tumors by upregulating CCL2 expression. Mechanistically, TJP1 interacted with TWIST1 and enhanced the transcriptional activity of CCL2. The impairment of tumor angiogenesis caused by knockdown of TJP1 was dramatically rescued by overexpression of TWIST1. Furthermore, TJP1 recruited USP2, which deubiquitinated TWIST1, thereby protecting TWIST1 from proteasome-mediated protein degradation. In conclusion, our results suggest that TJP1 controls angiogenesis in BLCA via TWIST1-dependent regulation of CCL2. We demonstrate that TJP1 functions as a scaffold for the interaction between USP2 and TWIST1 and this may provide potential therapeutic targets in bladder cancer.

Objective: The purpose of this study was to observe the effect of hyperocclusion on the remodeling of gingival tissues and detect the related signaling pathways. Design: Hyperocclusion models were established by tooth extraction in mice. The mice were sacrificed at 3, 7, 14, 28, or 56 days after the surgery, and the left mandibular first molars with gingival tissues were isolated and examinations were focused on the gingival tissues. Apoptotic cells were examined using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technology. Proliferating cells, p65, inflammatory cytokines, and beta-catenin were detected using immunohistochemical methods. Results: A series of apoptosis and proliferation responses were triggered in stressed gingival tissues. It was observed that the levels of p65, proinflammatory factors including interleukin-1 beta and tumor necrosis factor-alpha in extraction group were higher compared with those from mice with intact dentition, and peaked on days 14, 14 and 7 respectively. The expression of beta-catenin was increased under hyperocclusion situations, peaked on day 14, and declined to the initial levels over time. Conclusions: The results of this study suggest that hyperocclusion causes remodeling of the gingival tissues by activating a series of adaptive responses. Both nuclear factor kappa B and Wnt/beta-catenin signaling pathways may be responsible for those adaptive responses though the exact mechanism is not clear.

N, N-Dimethylformamide (DMF) can cause liver damage in occupationally exposed workers, but the molecular mechanism of DMF-induced liver damage has not been fully elucidated. Researches have proved that lncRNA plays a major function in chemical-induced liver toxicity and can be used as a biomarker and therapeutic target for liver injury. In order to verify that lncRNA also participates in DMF-induced liver damage, we treated HL-7702 cells with 75 or 150 mM DMF, and obtained lncRNA expression profiles through high-throughput sequencing. Among the differentially expressed lncRNAs, lncRNA SNHG12 was proved to be significantly downregulated in DMF-treated HL-7702 cells and participate in DMF-mediated apoptosis, even under long-term low-dose DMF exposure (5-10 mM, 8 weeks). In addition, according to bioinformatics analysis, miR-218-5p is expected to be a potential target of SNHG12, which was verified by the dual luciferase reporter assay in HEK293FT cells. MiR-218-5p mimic can induce apoptosis in HL-7702 cells. Among the predicted targets of miR-218-5p, protein kinase C epsilon (PRKCE) was reported to be involved in apoptosis, and was indeed downregulated by miR-218-5p mimic in our study. Further experiments showed that changes of the expression of SNHG12 can affect the expression of PRKCE. In the epidemiological study of occupational population, we also found that SNHG12 was downregulated in the serum exosomes of workers exposed to DMF. These results indicated that SNHG12 can mediate DMF-induced apoptosis of HL-7702 cells through miR-218-5p/PRKCE pathway.

N,N-dimethylformamide (DMF) is an organic compound widely used in industrial production processes as a solvent with a low evaporation rate. Excessive exposure to DMF may lead to liver damage. Oxidative stress has been reported as one of the main causes of DMF-induced hepatotoxicity. Several doses of DMF (0, 1, 5, and 10 mM) were used to treat HL-7702 cells for a relatively long period to simulate the actual exposure pattern in occupational settings, and oxidative stress was induced. Previous studies illustrated that circular RNA (circRNA) plays a vital role in sustaining hepatocyte physiological function. To explore whether aberrant circRNA expression is involved in DMF-induced excessive ROS generation and hepatotoxicity, high-throughput transcriptional sequencing was performed to identify the altered circRNA expression profiles in HL-7702 liver cells after treatment with 0, 75, or 150 mM DMF for 48 h. We found that levels of induced oxidative stress were similar to those in the long-term exposure model. Among the altered circRNAs, one circRNA (hsa_circ_0005915) was significantly upregulated after DMF exposure, and it affected DMF-mediated oxidative stress in HL-7702 cells. Further experiments revealed that hsa_circ_0005915 downregulated the expression of nuclear factor erythoid-2-related factor 2 (NRF2) at the post-transcriptional level via promoting the ubiquitination and degradation of NRF2, which led to the increase of ROS accumulation. Further investigation demonstrated that the expression levels of NRF2-regulated antioxidative genes—heme oxygenase 1 (HO1) and NAD(P)H quinone dehydrogenase 1 (NQO1)—indeed declined after the overexpression of hsa_circ_0005915. In vivo study also indicated that DMF exposure can upregulate the expression of mmu_circ_0007941 (homologous circRNA of hsa_circ_0005915) and downregulated Nrf2 and Ho1 proteins. In summary, our results revealed that hsa_circ_0005915 plays an important role in promoting DMF-induced oxidative stress by inhibiting the transcriptional activity of the NRF2/ARE axis, which provides a potential molecular mechanism of DMF-mediated hepatotoxicity.

Primary tumor (PT) and metastatic lymph node (MLN) status have a great influence on diagnosis and treatment of lung cancer. Our main purpose was to investigate the imaging characteristics of PT or MLN by applying the 18F-FDG PET dynamic modeling approach for non-small cell lung cancer (NSCLC). Dynamic 18F-FDG PET scans were performed for 76 lung cancer patients, and 62 NSCLC cases were finally included in this study: 37 with newly diagnosed early and locally advanced lung cancer without distant metastases (group M0) and 25 metastatic lung cancer (group M1). Patlak graphic analysis (Ki calculation) based on the dynamic modeling and SUV analysis from conventional static data were performed. For PT, both KiPT (0.050 ± 0.005 vs 0.026 ± 0.004 min−1, p < 0.001) and SUVPT (8.41 ± 0.64 vs 5.23 ± 0.73, p < 0.01) showed significant higher values in group M1 than M0. For MLN, KiMLN showed significant higher values in M1 than M0 (0.033 ± 0.005 vs 0.016 ± 0.003 min−1, p < 0.01), while no significant differences were found for SUVMLN between M0 and M1 (4.22 ± 0.49 vs 5.57 ± 0.59, p > 0.05). Both SUV PT and KiPT showed significant high values in squamous cell carcinoma than adenocarcinoma, but neither SUVPT nor KiPT showed significant differences between EGFR mutants versus wild types. The overall Spearman analysis for SUV and Ki from different groups showed variable correlation (r = 0.46–0.94). The dynamic modeling for MLN (KiMLN) showed more sensitive than the static analysis (SUV) to detect metastatic lymph nodes in NSCLC, although both methods were sensitive for PT. This methodology of non-invasive imaging may become an important tool to evaluate MLN and PT status for patients who cannot undergo histological examination. The clinical trial registration number is NCT03679936 (http://www.clinicaltrials.gov/).

Excessive exposure toN,N-dimethylformamide (DMF) can lead to occupational liver poisoning in workers; however, the underlying mechanism is not fully clarified. The importance of microRNAs (miRNAs) in chemical-induced hepatotoxicity has been demonstrated. To determine whether miRNAs are also involved in DMF-induced hepatotoxicity, we systematically analyzed the miRNA expression profiles in DMF-treated (75 and 150 mm) HL-7702 liver cells and controls by high-throughput sequencing. Among the altered miRNAs, miR-192-5p was the most significantly upregulated in HL-7702 cells after DMF exposure and was involved in DMF-mediated cell apoptosis. By contrast, suppression of miR-192-5p in HL-7702 cells attenuated the apoptosis induced by DMF. Furthermore, the anti-apoptotic gene (NIN1/RPN12 binding protein 1 homolog [NOB1]) was predicted to be a potential miR-192-5p target according to bioinformatics analysis. The direct interaction between miR-192-5p and NOB1 was confirmed by the dual-luciferase activity assay in HEK293FT cells. Overexpression of miR-192-5p efficiently reduced NOB1 mRNA and protein expression in HL-7702 cells. Alteration in NOB1 expression influenced DMF-induced hepatotoxicity by affecting hepatic apoptosis. In addition, the inverse correlation between miR-192-5p expression levels and NOB1 expression was further confirmed in DMF-exposed mouse liver tissue samples. These observations demonstrated that promotion of apoptosis from the suppression of NOB1 by miR-192-5p overexpression was responsible for the DMF-induced hepatotoxicity. This work provides the molecular mechanism at the miRNA level for hepatic apoptosis induced by DMF.

N , N ‐dimethylformamide (DMF) is a colorless industrial solvent that is frequently used for chemical reactions. Epidemiologic studies and clinical case reports have consistently indicated that the main toxic effect after exposure to DMF is hepatotoxicity. Previous studies have suggested that oxidative stress is the pivotal molecular event of DMF‐mediated hepatotoxicity; however, its underlying mechanism remains unclear. In this study, we found that DMF (0–150 mM) exposure induced an increase in reactive oxygen species (ROS) levels and inhibited the transcriptional activity of nuclear factor erythroid‐2‐related factor‐2 (NRF2) in a dose‐dependent manner. Subsequently, our research revealed that the elevated ROS levels and the decline in NRF2‐mediated anti‐oxidative response in HL‐7702 and HuH6 cells might be due to the DMF‐induced accumulation of retinoid X receptor α (RXRα) protein. Further investigation demonstrated that phosphorylation of the RXRα protein, which is mediated by the activation of extracellular signal‐regulated kinase (ERK), leads to the inhibition of RXRα protein degradation and in turn the accumulation of RXRα after DMF exposure. These findings provide information that improves our understanding of the role of RXRα in DMF‐induced hepatotoxicity.

Purpose/Aim of the study: Interleukin (IL)-35 is a newly identified IL-12 cytokine family member and reveals immunosuppressive activity to CD8+ T cells in inflammation, infectious diseases, and cancers. However, little is known regarding IL-35 function in osteosarcoma. Thus, the aim of the current study was to investigate the regulatory function of IL-35 to CD8+ T cells in osteosarcoma. Materials and methods: Thirty-five osteosarcoma patients and 20 healthy individuals were enrolled. Serum CD4+CD25+CD127dim/- regulatory T cells (Tregs) and CD8+ T cells were purified. IL-35 concentration in serum and cultured supernatants was measured by enzyme-linked immunosorbent assay. Osteosarcoma cell line MG-63 cells and CD8+ T cells were stimulated with recombinant IL-35 in vitro, and modulatory function of IL-35 on these cells was assessed by investigation of cellular proliferation, cell cycle, apoptosis, and cytokine production. Results: Serum IL-35 and Treg-secreting IL-35 were significantly elevated in osteosarcoma patients. IL-35 stimulation did not affect proliferation, apoptosis, or cell cycle of MG-63 cells. Purified peripheral CD8+ T cells from osteosarcoma patients revealed dysfunctional property, which presented as decreased mRNA expressions for perforin, granzyme B, and granulysin, as well as reduced cytolytic (direct lysis of target MG-63 cells) and noncytolytic (interferon-γ and tumor necrosis factor-α production) function in coculture systems. Moreover, IL-35 stimulation further diminished cytolytic and noncytolytic activity of CD8+ T cells from osteosarcoma patients. Conclusions: The current data indicated that IL-35 contributed to CD8+ T-cell dysfunction and limited antitumor immune response in osteosarcoma.

本实验以S 100A9蛋白和星形胶质细胞为原材料,采用CCK-8法检测不同浓度S100A9对星形胶质细胞的增殖抑制效应以及检测FPS-ZM1、TAK-242对S100A9作用于星形胶质细胞后的保护作用;应用流式细胞术检测细胞凋亡变化;采用ELISA法检测S100A9作用于细胞后上清液中IL-1、IL-6和TNF-α 的水平;使用Western blotting检测细胞表达JNK、P38-MAPK、Bcl-2蛋白浓度水平的变化.研究发现,与对照组相比,0.5 mg/mLS 100A9作用24h后,星形胶质细胞出现显著的增殖抑制效应,且凋亡率显著增高(p＜0.01).S100A9+TAK-242组、S100A9+FPS-ZM1 +TAK-242组作用24h后细胞增殖抑制显著降低且凋亡率显著降低(p＜0.01),S 100A9+FPS-ZM1组作用后凋亡率与S100A9组无显著差异(p＜0.05).与对照组相比,S100A9单独作用24h后星形胶质细胞分泌IL-1、IL-6和TNF-α显著增加(p＜0.01),S 100A9+FPS-ZM1组、S100A9+TAK-242组、S100A9+FPS-ZM1 +TAK-242组,上述因子无明显变化.与对照组相比,S100A9组JNK、P38-MAPK蛋白浓度升高,而Bcl-2蛋白浓度降低;S100A9+TAK-242组、S100A9+FPS-ZM1 +TAK-242组JNK、P38-MAPK蛋白浓度均降低,Bcl-2蛋白浓度升高(p＜0.05);S 100A9+FPS-ZM1组3种蛋白浓度变化与S100A9组无明显差异.综上表明,S100A9可能通过上调JNK、P38-MAPK及下调Bcl-2蛋白表达导致细胞凋亡从而抑制其增殖.TLR-4受体的特异性抑制剂TAK-242减轻了S100A9促凋亡作用,表明S100A9可能通过结合星形胶质细胞膜上TLR-4受体促进凋亡作用.S100A9通过结合细胞膜上TLR-4受体和RAGE受体刺激细胞炎性因子产生增多.

Background: Activation of N-methyl-D-aspartate (NMDA) receptor by reactive oxygen species (ROS) in the spinal cord plays an important role in the development of hyperalgesia in several neuropathic pain models. The study examined the involvement of ROS-NMDA signaling pathway in remifentanil-induced postoperative hyperalgesia.Methods: Nociceptive responses were measured by paw withdrawal mechanical threshold (PWT) and paw withdrawal thermal latency (PWL) before and up to day 5 after remifentanil infusion. Spinal delivery of MitoSOX red was performed to detect mitochondria( ROS. Changes in expression of NMDA receptor subunits (NR1 and NR2B) in the spinal cord were analyzed by immunofluorescence and Western blotting. Intraperitoneal injection of phenyl-N-tert-butylnitrone (PBN), a non-selective ROS scavenger, was administrated to investigate the role of ROS in remifentanil-induced postoperative hyperalgesia.Results: Intraoperative infusion of remifentanil induced postoperative mechanical allodynia and thermal hyperalgesia. ROS production, phosphorylated NR1 and NR2B subunits of NMDA receptor were found to be significantly increased in the spinal dorsal horn after intraoperative remifentanil infusion. However, remifentanil-induced postoperative hyperalgesia was suppressed by pretreatment of PBN. In addition, reduction of ROS by PBN prevented enhanced phosphorylation of NR1 and NR2B subunits.Conclusion: These findings indicated that ROS-dependent activation of NMDA receptor in the spinal cord might be a potential mechanism underlying remifentanil-induced postoperative hyperalgesia. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

PURPOSE:Long-term topical antiglaucoma therapy is considered a significant risk factor for failure of trabeculectomy. We investigated the effects of antiglaucoma drugs on proinflammatory cytokine and chemokine release from cultured human Tenon's capsule fibroblasts (HTFs) as well as the signaling pathways that underlie such effects.MATERIALS AND METHODS:Release of the proinflammatory cytokine interleukin (IL)-6 and the chemokines IL-8 and monocyte chemotactic protein (MCP)-1 was measured with enzyme-linked immunosorbent assays. The phosphorylation (activation) of mitogen-activated protein kinases (MAPKs) as well as the phosphorylation and degradation of the nuclear factor-κB (NF-κB) inhibitor IκB-α were assessed by immunoblot analysis.RESULTS:Latanoprost stimulated the release of IL-6, IL-8, and MCP-1 from HTFs in a concentration-dependent and time-dependent manner, whereas timolol maleate and pilocarpine had no such effects. Latanoprost also activated the MAPKs extracellular signal-regulated kinase, p38, and c-Jun NH2-terminal kinase as well as induced the phosphorylation and degradation of IκB-α in these cells. The latanoprost-induced release of IL-6, IL-8, and MCP-1 was attenuated by inhibitors of MAPK (PD98059, SB203580, or JNK inhibitor II) or NF-κB (IκB kinase 2 inhibitor) signaling pathways.CONCLUSIONS:Latanoprost induced proinflammatory cytokine and chemokine release from HTFs in a manner dependent on MAPK and NF-κB signaling. These effects of latanoprost might influence bleb scarring after filtration surgery.

Purpose: Matrix metalloproteinases (MMPs) mediate the degradation of extracellular matrix proteins and are implicated in the pathogenesis of corneal ulceration. Tranilast, a clinically approved antiallergy drug, has been found to exert various anti-inflammatory effects. We examined the effects of this agent on MMP expression in cultured corneal fibroblasts.Methods: Human corneal fibroblasts were cultured in the absence or presence of interleukin-1 beta (IL-1 beta) or tranilast. The release of MMPs into culture supernatants was assessed by immunoblot analysis and gelatin zymography, and the cellular abundance of MMP mRNAs was determined by reverse transcription and real-time polymerase chain reaction analysis. The phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappa B (NF-kappa B) inhibitor I kappa B-alpha was examined by immunoblot analysis.Results: The IL-1 beta-induced expression of MMP-1, -2, and -3 in corneal fibroblasts was inhibited by tranilast in a concentration-and time-dependent manner. It was also attenuated by synthetic inhibitors of MAPK or NF-kappa B signaling pathways. Tranilast inhibited the IL-1 beta-induced phosphorylation of the MAPKs extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK) as well as the phosphorylation and degradation of I kappa B-alpha. Tranilast did not exhibit cytotoxicity for corneal fibroblasts.Conclusions: Tranilast inhibits the IL-1 beta-induced production of MMP-1, -2, and -3 by human corneal fibroblasts, with this action likely being mediated through suppression of MAPK and NF-kappa B signaling pathways. Tranilast thus warrants further investigation as a potential treatment for corneal ulceration on the basis of its inhibition of MMP expression in corneal fibroblasts.