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Background Although long noncoding RNA HLA complex group 18 (lncRNA HCG18) has been suggested to regulate cell growth in several tumours, the function of HCG18 in epithelial ovarian cancer (EOC) and its mechanism are still unclear. Methods shRNAs were applied to reduce HCG18 and related genes. For overexpression of miRNA, a miRNA mimic was transfected into cells. Quantitative real-time PCR (qRT–PCR) was used to detect levels of HCG18, miR-29a/b, and mRNAs. MTT, colony formation, wound healing and Transwell assays were used to evaluate cell proliferation, migration and invasion, respectively. A luciferase reporter assay was utilized to evaluate NF-κB activity and the binding of miRNAs with HCG18 or TRAF4/5. BALB nude mice injected with cells stably expressing shHCG18 or shNC were used for in vivo modelling. Subcutaneous tumour growth was monitored in nude mice, and immunohistochemistry (IHC) was used to determine expression of the proliferation marker Ki67. Results Abnormal expression of HCG18 and miR-29a/b was observed in EOC tissues. Knockdown of HCG18 using shRNA inhibited proliferation, migration, EMT and the proinflammatory pathway in EOC cells. miR-29a/b mimics and TRAF4/5 knockdown exhibited effects similar to HCG18 knockdown. Further experiments suggested that HCG18 directly targets miR-29a/b and upregulates TRAF4/5 expression, which are inhibited by targeting miR-29a/b. Moreover, overexpression of TRAF4/5 antagonized the inhibitory effect of HCG18 knockdown, suggesting that they are involved in HCG18-mediated oncogenic effects. Silencing HCG18 reduced tumour size and levels of Ki67 and TRAF4/5 while increasing miR-29a/b levels in vivo. Conclusions Taken together, our data revealed an oncogenic signalling pathway mediated by HCG18 in ovarian cell lines, which functions as a ceRNA of miR-29a/b and thus derepresses expression levels of TRAF4/5, facilitating NF-κB pathway-mediated promotion of EOC cell proliferation and migration.

TNF receptor-associated factors (TRAFs) function as intracellular adaptor proteins utilized by members of the TNF receptor superfamily, such as CD40. Among the TRAF family proteins, TRAF5 has been identified as a potential regulator of CD40. However, it remains unclear whether TRAF5 regulates the generation of germinal center (GC) B cells and antigen-specific antibody production in the T-dependent (TD) immune response. TRAF5-deficient (Traf5−/−) and TRAF5-sufficient (Traf5+/+) mice were immunized in the footpad with 2,4,6-trinitrophenol-conjugated keyhole limpet hemocyanin (TNP-KLH) and complete Freund’s adjuvant (CFA). We found that GC B cell generation and antigen-specific IgM and IgG1 production were significantly impaired in Traf5−/− mice compared to Traf5+/+ mice. The expression levels of CD40-target genes Fas and Lta, which are involved in GC formation, were significantly decreased in B220+ cells isolated from immunized Traf5−/− mice. Traf5−/− B cells showed decreased antibody production, proliferation, and induction of CD40-target genes Tnfaip3, Tnfsf4, and Cd80 in response to agonistic Fc-CD40L protein in vitro. Furthermore, administration of TNP-KLH and Fc-CD40L to Traf5−/− mice resulted in a severe loss of GC B cell development. These results highlight the crucial role of TRAF5 in driving CD40-mediated TD immune response in vivo.

Hepatocellular carcinoma (HCC) is a common malignancy with poor prognosis and high mortality. This study aimed to explore the oncogenic mechanisms of TRAF5 in HCC and provide a novel therapeutic strategy for HCC. Human HCC cell lines (HepG2, HuH7, SMMC-LM3, and Hep3B), normal adult liver epithelial cells (THLE-2), and human embryonic kidney cells (HEK293T) were utilized. Cell transfection was performed for functional investigation. qRT-PCR and western blotting were used to detect mRNA expression of TRAF5, LTBR, and NF-κB and protein expression of TRAF5, p-RIP1(S166)/RIP1, p-MLKL(S345)/MLKL, LTBR, and p-NF-κB/NF-κB. Cell viability, proliferation, migration, and invasion were evaluated using CCK-8, colony formation, wound healing, and Transwell assays. Cell survival, necrosis, and apoptosis were assessed using flow cytometry and Hoechst 33342/PI double staining. Co-immunoprecipitation and immunofluorescence were performed to determine the interaction between TRAF5 and LTBR. A xenograft model was established to validate the role of TRAF5 in HCC. TRAF5 knockdown inhibited HCC cell viability, colony formation, migration, invasion, and survival but enhanced necroptosis. Additionally, TRAF5 is correlated with LTBR and TRAF5 silencing down-regulated LTBR in HCC cells. LTBR knockdown inhibited HCC cell viability, while LTBR overexpression eliminated the effects of TRAF5 deficiency on inhibiting HCC cell proliferation, migration, invasion, and survival. LTBR overexpression abolished the promotive function of TRAF5 knockdown on cell necroptosis. LTBR overexpression undid the suppressive effect of TRAF5 knockdown on NF-κB signaling in HCC cells. Moreover, TRAF5 knockdown suppressed xenograft tumor growth, inhibited cell proliferation, and promoted tumor cell apoptosis. TRAF5 deficiency facilitates necroptosis in HCC by suppressing LTBR-mediated NF-κB signaling.

Colorectal cancer is one of the most common malignant tumors worldwide, significantly impacting human health. Cantharidin (CTD), an active compound derived from the Spanish fly, exhibits antitumor properties. Its derivative, norcantharidin (NCTD), is synthesized by removing methyl groups from positions 1 and 2 of cantharidin. NCTD has demonstrated lower toxicity while maintaining similar antitumor effects compared to CTD. However, the mechanism by which NCTD exerts its effects against colorectal cancer remains unclear. Here, we conducted a comprehensive analysis of the effects of NCTD on colorectal cancer both in vitro and in vivo. Whole-transcriptome sequencing and bioinformatics tools were employed to identify potential key targets of NCTD in the treatment of colorectal cancer. Additionally, we designed folate-receptor-targeting NCTD liposomes (FA-NCTD) and assessed their anticancer efficacy in vivo. NCTD effectively inhibited cell viability, clonal formation, and migration in HCT116 and HT-29 cell lines. NCTD also induced apoptosis, influenced the cell cycle, altered mitochondrial membrane potential, and increased reactive oxygen species levels. The whole-transcriptome sequencing and bioinformatics analysis identified TRAF5 as a key target for NCTD’s action against colorectal cancer. Furthermore, NCTD was found to regulate the TRAF5/NF-κB signaling pathway in both HCT116 and HT-29 cells. The FA-NCTD liposomes demonstrated effective tumor targeting and significantly inhibited tumor growth in vivo. This result showed that NCTD effectively suppresses the malignant proliferation of colon cancer cells by modulating the TRAF5/NF-κB signaling pathway and inducing programmed apoptosis, thereby offering a novel strategy for colorectal cancer treatment. The prepared FA-NCTD liposomes provide a promising approach for achieving the precise targeting and controlled release of NCTD.

The modulation of leptin sensitivity in hypothalamic neurons plays a crucial role in metabolic regulation and the development of obesity. Three distinct approaches, exposure to leptin (LEPA), administration of leptin antagonist (LANTA), and treatment with palmitate (PA), were explored in this study to assess their effects on adult-derived mHypoA-2/12 neurons and the resulting transcriptomic signatures. To this end, 3’ mRNA-Seq transcriptome analysis was employed, unexpectedly revealing downregulation of key genes associated with the NOD-like receptor signaling pathway ( Irf9 , Mapk3 , Stat2 , Nfkbia , Ikbkg , Rela , Cxcl1 , and Traf5 ), the C-type lectin receptor signaling pathway ( Nfkb2 , Irf9 , Mapk3 , Stat2 , Nfkbia , Ikbkg , Rela , and Ptgs2 ), the NF kappa B signaling pathway ( Nfkbia , Ikbkg , Nfkb2 , Rela , Traf5 , Cxcl1 , and Ptgs2 ), and the IL 17 signaling pathway ( Nfkbia , Ikbkg , Mapk3 , Rela , Traf5 , Cxcl1 , and Ptgs2 ). These findings help elucidate the molecular mechanisms through which these factors influence leptin sensitivity and provide insights into the pathways implicated in the development of leptin resistance in hypothalamic neurons. The surprising downregulation of these pathways suggests a complex interplay between leptin signaling and the cellular stress response in hypothalamic neurons. This alteration may reflect adaptive mechanisms in response to prolonged leptin or fatty acid exposure. Understanding these dynamics is essential for elucidating the role of hypothalamic inflammation in the progression of leptin resistance and associated metabolic disorders.

ObjectivesIntimal hyperplasia is a serious clinical problem associated with the failure of therapeutic methods in multiple atherosclerosis-related coronary heart diseases, which are initiated and aggravated by the polarization of infiltrating macrophages. The present study aimed to determine the effect and underlying mechanism by which tumor necrosis factor receptor-associated factor 5 (TRAF5) regulates macrophage polarization during intimal hyperplasia.MethodsTRAF5 expression was detected in mouse carotid arteries subjected to wire injury. Bone marrow-derived macrophages, mouse peritoneal macrophages and human myeloid leukemia mononuclear cells were also used to test the expression of TRAF5 in vitro. Bone marrow-derived macrophages upon to LPS or IL-4 stimulation were performed to examine the effect of TRAF5 on macrophage polarization. TRAF5-knockout mice were used to evaluate the effect of TRAF5 on intimal hyperplasia.ResultsTRAF5 expression gradually decreased during neointima formation in carotid arteries in a time-dependent manner. In addition, the results showed that TRAF5 expression was reduced in classically polarized macrophages (M1) subjected to LPS stimulation but was increased in alternatively polarized macrophages (M2) in response to IL-4 administration, and these changes were demonstrated in three different types of macrophages. An in vitro loss-of-function study with TRAF5 knockdown plasmids or TRAF5-knockout mice revealed high expression of markers associated with M1 macrophages and reduced expression of genes related to M2 macrophages. Subsequently, we incubated vascular smooth muscle cells with conditioned medium of polarized macrophages in which TRAF5 expression had been downregulated or ablated, which promoted the proliferation, migration and dedifferentiation of VSMCs. Mechanistically, TRAF5 knockdown inhibited the activation of anti-inflammatory M2 macrophages by directly inhibiting PPARγ expression. More importantly, TRAF5-deficient mice showed significantly aggressive intimal hyperplasia.ConclusionsCollectively, this evidence reveals an important role of TRAF5 in the development of intimal hyperplasia through the regulation of macrophage polarization, which provides a promising target for arterial restenosis-related disease management.

MicroRNA-873 (miR-873) has been reported to be dysregulated in a variety of malignancies, however, the biological function and underlying molecular mechanism of miR-873 in colorectal cancer (CRC) remain unclear. In the present study we found that the expression levels of miR-873 were markedly decreased in CRC cell lines and tissues from patients. Statistical analysis revealed that miR-873 expression was inversely correlated with the disease stage of CRC. Kaplan-Meier survival analysis revealed that patients with CRC with lower miR-873 expression had shorter overall survival rates. Additionally, downregulation of miR-873 enhanced the proliferation of CRC cells, while upregulation of miR-873 reduced this proliferation. Furthermore, we found that tumor necrosis factor (TNF) receptor-associated factor 5 (TRAF5) and TGF-β activated kinase 1 (MAP3K7) binding protein 1 (TAB1) were direct targets of miR-873 in CRC cells. A luciferase assay revealed that ectopic expression of miR-873 significantly reduced nuclear factor κB (NF-κB) luciferase activity, while ectopic expression of miR-873 inhibitor enhanced luciferase activity, suggesting that downregulation of miR-873 can activate NF-κB signaling. Therefore, our findings established a tumor-suppressive role for miR-873 in the inhibition of CRC progression, which may be employed as a novel prognostic marker and as an effective therapeutic target for CRC.

This study aims to figure out the mechanism of astragaloside IV (AS-IV) in the protection of podocyte apoptosis in diabetic nephropathy (DN) rats. Streptozotocin (STZ) was used to induce diabetes in rats, and the diabetic rats were treated with 5 mg/kg/d of AS-IV for 12 weeks. Albuminuria level, relative TUG1 and TRAF5 levels, and TRAF5 and cleaved-caspase-3 protein levels were examined by ELISA, quantitative reverse transcription (qRT)-PCR, and Western blot analyses, respectively. The interaction between TUG1 and TRAF5 was confirmed by RNA pull-down and RNA precipitation. TUNEL assay was used to detect podocyte apoptosis. Compared with control rats, DN rats had higher albuminuria and TRAF5 levels and lower TUG1 level. AS-IV treatment attenuated albuminuria and TRAF5 levels and improved TUG1 level in DN rats. TUG1 was downregulated and TRAF5 was upregulated in high-glucose-treated MPC5 cells, and AS-IV ameliorated the TUG1 level. In addition, TUG1 interacted with TRAF5, and TUG1 overexpression promoted degradation of TRAF5 protein. Besides, AS-IV modulated TRAF5 expression through regulating TUG1. AS-IV decreased podocyte apoptosis via the TUG1/TRAF5 pathway. Finally, in vivo experiment proved that si-TUG1 abrogated the protective effect of AS-IV on DN. AS-IV attenuated podocyte apoptosis and protected diabetic rats from DN via the lncRNA-TUG1/TRAF5 pathway.

Numerous lines of evidence have shown that microRNAs (miRNAs) play a vital role in regulating the progression in many types of cancers, including T cell acute lymphoblastic leukemia (T-ALL). In this study, the potential underlying mechanism and functional role of miR-141-3p in T-ALL cells were determined. We found that the expression level of miR-141-3p was significantly downregulated, while that of tumor necrosis factor receptor-associated factor 5 (TRAF5) was strongly upregulated in tissues from patients with T-ALL compared with healthy controls. Subsequently, upregulation of miR-141-3p significantly repressed T-ALL cell proliferation and promoted cell apoptosis. Conversely, downregulation of miR-141-3p significantly inhibited cell apoptosis and enhanced T-ALL cell proliferation. We also verified that TRAF5 was the direct target of miR-141-3p in T-ALL cells. Additionally, TRAF5 overexpression significantly repressed cell apoptosis and increased T-ALL cell proliferation. In summary, miR-141-3p regulates T-ALL cell progression by directly targeting TRAF5, and may serve as a potential therapeutic target for T-ALL.

Biliary atresia (BA) is an obliterating fibrous inflammatory bile duct disease in infants. Interleukin 17A (IL-17A) is abnormally expressed in patients with BA; however, the mechanism of its expression is unclear. Liver tissues from patients with BA and those with anicteric choledochal cysts (non-BA) were collected. The expression of genes and proteins was determined using RT-qPCR and western blot. Cell biological activities, including viability and proliferation, were evaluated by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2 -deoxyuridine (EdU) assay. Glucose uptake and lactate and ATP levels were examined using commercial kits. The extracellular acidification rate (ECAR) level was evaluated by the XF96 Extracellular Flux analyzer. The interactions among TRAF2, TRAF5, and human antigen R (HuR) were validated using co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down. In BA patients, IL-17A, TRAF2, TRAF5, and PFKFB3 were highly expressed, and IL-17A expression was positively correlated with PFKFB3, TRAF2, and TRAF5 expression, respectively. IL-17A elevated PFKFB3 expression and promoted glycolysis and the proliferation and fibrosis of hepatic stellate cells (HSCs), which were abolished by 2-deoxy-D-glucose (2-DG) and PFKFB3/TRAF2/TRAF5 silencing. Mechanistically, IL-17A promoted the interactions among HuR, TRAF2 and TRAF5 to form the TRAF2/TRAF5/HuR complex, thereby enhancing PFKFB3 expression. IL-17A facilitates glycolysis and HSC fibrosis by promoting TRAF2/TRAF5/HuR complex formation to regulate PFKFB3 expression.